Development of mass spectrometry based human cell secretome analytics: The T-lymphocyte as a model system

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human cell secretome analytics

The T-Iymphocyte

as a

model system

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human eell seeretome analyties

The T-Iymphocyte

as a

model system

PROEFSCHRIFf

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Delft .

Prorneth

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Delft

terverkrijging van de graad van doctor aan de Technische Universiteit Delft,

op gezag van de Rector Magnificus prof. ir. K.C.A.M. Luyben, voorzitter van het College voor Promoties,

in het openbaar te verdedigen op 14 december 2011 om 12:30uur door

Inez FINOULST

Industrieel Ingenieur Chemie optie Biochemie - Karel de Grote Hogeschool geboren te Wilrijk, België.

Dit proefschrift is goedgekeurd door de promotor: Prof.dr. P. D.E.M. Verhaert

Samenstelling promotiecomissie:

Rector Magnificus voorzitter

Prof.dr.P.D.E.M.Verhaert Delft University of Technology, promotor Prof.dr.

J

.H. de Winde Delft University of Technology

Prof. dr.ir.

J

.J.

Heijnen Delft University of Technology Prof. dr.P. Andrén Uppsala University, Sweden Prof.dr. G.Corthals University of Turku, Finland Dr.M.W.H. Pinkse Delft University of Technology

Dr.E. Bos MSD

Prof. dr.W.R.Hagen Delft University of Technology, reservelid

This research was financially supported by European Marie Curie Research Training Network grant number MCRTN-CT-2006-035854.

Copyright©2011 by Inez Finoulst

Printed and bound in The Netherlands by CPI Wörmann Print Service.

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Table of Contents

Summary

Samenvatting

Chapter1 General introduetion

vii

ix xiii

1

Chapter 2 Samplepreparationtechniques for the untargetedLC-MS based discovery of pep-tides in complex biologica I matrices 15 Chapter3 Development of a mass speetrometry based method for the detection and

identifi-cation of proteins secretedby cells in culture 35 Chapter4 Identification of low abundant secreted proteins and peptides from primary

cul-ture supematants of human T-cells 49 Chapter5 Non-targeted LC MS/MS analysis of primary human T-eell culture supernatants

detects activation dependent secreted proteins/peptides 67 Chapter 6 Evaluation of isobaric tandem mass tags for quantitative proteomics and

pep-tidomics 85

Chapter7 Concluding remarks and future perspectives

Bibliography

Appendix

Curriculum vitae Publications Dankwoord 101 107 127 149 151 155

The objective of this thesis was to develop a method for the comprehensive analysis of pep-tides and small proteins secreted by living cells. This involved the evaluation and innova-tive combination of many aspects of recent proteomics and peptidomics analytics to allow detection, identification and, in a last step, quantitation of this biologically relevant subset of the cellular proteome/peptidome/secretome. T-cells were chosen as model system be-cause of well-established aspects of their secretory biology as weil as the large present-day biomedical interest in them. T-cells secrete various known cytokines, but immunologists presume more secretory peptides still to be discovered. Specific conditions are known un-der which T-cells secrete such peptides: activation of the T-cell receptor triggers the release of interleukin-2 (IL-2, a 16 kDa peptide). This work faced various challenges that were tack-led:

First of all it is imperative, when studying material of human (donor) origin, that the analyt-ics employed/developed have sufficient sensitivity to allow access to the relevant analytes from the typically small sizes inherent to clinical samples. Following clinical sample collec-tion, cells need to be cultured in a way that enables both relevant biological experimentacollec-tion, as weil as appropriate proteome/peptidome/secretome analyses. T-cells are mammalian cells, requiring different culture conditions/media compared to microbial celis, which can grow on a relatively poor medium (so-called minimal medium). Minimal medium only contains salts and a very small amount of vitamins and sugar, but mammalian celis do not survive on it. The latter require so-called 'rich' medium, containing a variety of vitamins and (peptide/protein) growth factors. This is usually provided in the form of fetal bovine serum (FBS). The many proteins present in the added FBS, however, seriously interfere with the mass spectrometric (MS) analysis of acell's secretome. The analysis method generally can-not discrirninate between extemally added and secreted proteins and peptides, the latter of which are typically present in much lower concentrations than the abundant FBS proteins. Techniques such as ultrafiltratien or immunodepletion are commonly used for reducing

sample complexity (removing large size proteins. while leaving smaller peptides for analy-sis) and targeted removal of known abundant proteins. However, as low abundant peptides and small proteins are known to be transported by bigger and highly abundant 'carrier' proteins such as albumin, we have attempted to refrain from these techniques, because of the risk to lose peptides and proteins of interest during sample preparation/cleanup.

We evaluated two approaches for analysis and identification of the T-cell secretome. The first approach (Chapter 3) consists of a protocol in which separation of the proteins by gel-electrophoresis is followed by in-gel digestion before analysis by nanoLC-MS/MS. Whereas immuno-assay showed IL-2 was present in the cell culture supernatant, th is protocol did not allow IL-2 detection by mass spectrometry. The next 2 chapters (Chapter 4, Chapter 5) eva lu-ate the use of multidimensionalliquid chromatography before mass spectrometric analysis. By fractionating the samples first on a C4 reversed phase column and subsequently analyz-ing them by C18 nanoLC-MS/MS, IL-2 could be successfully identified in the cell culture supernatant of primary T-cells cultivated firstly in a defined medium, 'poor in added pro-teins' (Chapter 4), and finally also in conventional cell culture medium, i.e. supplemented with FBS (Chapter 5). Cultivating T-cells in FBS containing cell culture medium, keeps cells more viabie and allows more proteins to be detected and identified by the multidirnen-sional LC-MS/MS approach, compared to cells cultivated on serum free medium or defined medium. Among the proteins identified are several cytokines, and their presence in the su-pernatant was confirmed by conventional immuno-assays. Where Chapter 4 focuses on the development of the method, Chapter 5 also indicates how appropriate filtering of the data can facilitate interpretation.

With the previous 3 chapters quantifying proteins relying on a label-free approach, the last part of this thesis is considering protein quantitation approaches which make use of (iso-topic) labels. Chapter 6 illustrates how particularly isobaric labeling has been implemented in proteomics experiments in our laboratory. The use of 'Tandem Mass Tags' (TMT) for the quantitation of proteins and peptides is demonstrated for different species, sample types and biologicaI questions. Itwas initially applied to the neuroproteome of mouse forebrains (whole tissue extraction), a study in which conventional quantitation by 2-dimensional gel-electrophoresis was compared with TMT based quantitation. A next application of TMT concentrated on the secreted (neuro)peptidome, rather than on the whole neuroproteorne, from locally captured cockroaches. This project showed that TMT can be used to discover novel peptides that are involved in predefined biologicaI processes. The last TMT project mentioned deals with the quantitation of the full secreted proteome study of the bictechno-logically relevant fungus,Aspergillus niger.

In conclusion, this thesis reveals that by selecting a combination of multidirnensional liq-uid chromatography, state-of-the-art mass spectrometry and blo-informaties, the detection of small proteins and peptides secreted into a complex matrix becomes possible. The use of isobaric tags for quantitation allows for an accurate time-dependent evaluation of these se-creted molecules. With mass spectrometers constantly improving, it is only a matter of time before even lower abundant proteins and peptides can be detected, or sample preparation

Het doel van deze thesis was om een methode te ontwikkelen voor de uitvoerige analyse van peptides en kleine eiwitten gesecreteerd door levende cellen. Dit omvatte de evaluatie en vernieuwende combinatie van vele aspecten van recente proteomics en peptidomics anal-yses om detectie, identificatie en, in een laatste stap, kwantitatieve analyse van deze biolo-gisch relevante deelgroep van het cellulaire proteoom/peptidoom/secretoom toe te staan. T-cellen werden gekozen als modelsysteem omwille van de voldoende bewezen aspecten van hun secretorische biologie alsook vanwege de grote huidige biomedische interesse in deze cellen. T-cellen secreteren verschillende gekende cytokines, maar immunologen ver-moeden dat er nog meer secretorische peptides moeten ontdekt worden. De secretie van zulke peptides door T-cellen gebeurt onder specifieke condities:activa tie van de T-cel re-ceptor veroorzaakt de vrijgave van interleukine 2 (IL-2, een 16 kDa peptide). Het werk in deze thesis behandelt verschillende uitdagingen die werden aangegaan:

Ten eerste is het noodzakelijk bij het bestuderen van samples van menselijke (donor) orig-ine om analytische methoden te gebruiken/ontwikkelen die voldoende gevoelig zijn om detectie van de relevante moleculen in de typisch zeer kleine sample volumes, eigen aan klinische samples, toe te staan.Na het verzamelen van klinische samples, moeten de cellen zodanig in cultuur gebracht worden dat zowel relevante biologische experimenten als de geschikte proteoom/peptidoom/secretoom analyses mogelijk zijn. T-cellen zijn dierlijke cellen, die andere cultuur condities nodig hebben vergeleken met microbiële cellen. Deze laatste kunnen leven in een relatief arm medium (minimum medium), wat enkel zouten en een zeer kleine hoeveelhied vitamines en suiker bevat. Echter, dierlijke cellen kunnen hierin niet overleven, ze hebben een 'rijk' medium nodig wat een verscheidenheid aan vitamines en (peptide/proteïne) groeifactoren bevat. Dit wordt meestal in de vorm van fetaal bovien serum (FBS) toegevoegd. De vele eiwitten aanwezig in het toegevoegde FBS belemmeren echter de massa spectrometrische (MS) analyse van het celsecretoom. In het algemeen kan de analyse methode geen onderscheid maken tussen extern toegevoegde en door de cel

gesecreteerde proteïnes en peptides. Deze laatste zijn vaak aanwezig in aanzienlijk lagere concentraties dan de abundante FBS proteïnes. Technieken zoals ultrafiltratie of immun-odepletie worden algemeengebruiktvoor hetverminderenvansamplecomp lexiteit(grote proteïnes zonder de kleinere peptides verwijderen) en de gerichte verwijdering van gekende abundante eiwitten. Aangezien laag abundante peptides en kleine eiwitten door grotere en meer abundante'carrier ' eiwitten worden getransporteerd, hebben we echter getracht deze techn ieken te vermijden omwille van het risico om peptides en eiwitten van interesse te verliezen tijdens de sample voorbereiding.

Er werden 2 verschillende methodes voor analyse en identificatie van T-cel secretoom geëvalueerd. De eerste methode (Hoofdstuk 3) is een protocol waar eiwitten gescheiden worden door gel-electroforesegevolgd door in-gel digestie en analyse met nanoLC-MS/MS.

Waar immuno-assays de aanwezigheid van IL-2 in het celcultuur supernatant bevestigde, kon met dit protocol geen IL-2 geïdentificeerd worden met massa spectrometrie. De vol-gende 2 hoofdstukken (Hoofdstuk 4, Hoofdstuk5)evaluerenhet gebruik van

multidimen-sionele vloeistofchromatografie gecombineerd met massa spectrometrie. Door de samples eerst te fractioneren op een C4 reversed phase kolom en ze vervolgens te analyseren met C1S nanoLC-MS/MS kon IL-2 succesvol geïdentificeerd worden in het celcultuur super

-natant van primaire T-cellen gecultiveerd in een in eerste instantie gedefinieerd medium,

'a rm aan extern toegevoegde eiwitten' (Hoofdstuk 4), en vervolgens een standaard medium, i.e.medium met FBS toegevoegd (Hoofdstuk 5).

Cellen kweken in FBSbevattend celcultuur medium houdt de cellen meer leefbaar en laat toe meer eiwitten te detecteren en te identificeren met de multidimensionele LC-MS/MS

methode vergeleken met cellen gecultiveerd in serum vrij medium of gedefinieerd medium.

Tussen de geïdentificeerde eiwitten kunnen verschillende cytokines gevonden worden, en hun aanwezigheid werd bevestigd met traditioneleimrnuno-assays. Terwijl hoofdstuk 4 meer de nadruk legt op de ontwikkeling van de methode, maakt hoofdstuk5 duidelijk hoe geschikte datafiltering de interpretatie kan vergemakkelijken.

Terwijl de vorige3hoofdstukken voor het kwantificeren van de eiwitten gebruik maken van een label vrije methode, worden in het volgende hoofdstuk van deze thesis metho-den besproken voor kwantificeren met behulp van (isotopische) labels. Hoofdstuk 6 il-lustreert hoe isobarische labels voor proteomics experimenten in ons laboratorium wer-den geïmplementeerd. Het gebruik van 'Tandem Mass Tags' (TMT) voor kwantificatie van proteïnes en peptides wordt aangetoond voor verschillende species, sample types en biolo-gische vragen.In eerste instantie werd het gebruikt voor de analyse van het neuroproteoom van voorhersenen van de muis (volledige weefsel extractie), een studie die traditionele 2-dimensionele gel-electroforese vergelijkt met kwantificatie op basis van TMT labels. Een volgende toepassing van TMT concentreerde zich op het gesecreteerde (neuro)peptidoom,

in plaats van het volledige neuroproteoom, van lokaal gevangen kakkerlakken. Ditproject toonde aan dat TMT gebruikt kan worden voor de ontdekking van nieuwe peptides die in biologische processen betrokken zijn.Het laatste TMT project in dit hoofdstuk behandelt het kwantificeren van het volledige gesecreteerde proteoom van een biotechnologisch relevante

schimmel,Aspergillus niger.

Ter conclusie kan gesteld worden dat deze thesis duidelijk maakt dat door het com-bineren van multidimensionele vloeistofchromatografie, state-of-the-art massa spectrome-trie en bic-informatica, de detectie van kleine proteïnes en peptides gesecreteerd in een complexe matrix mogelijk wordt. Het gebruik van isobarische labels voor kwantificatie laat nauwkeurig tijdsafhankelijke evaluatie van deze gesecreteerde moleculen toe. Aangezien massa spectrometers continu evolueren en verbeteren, is het slechts een kwestie van tijd vooraleer nog minder abundante peptides kunnen gedetecteerd worden of dat sample voor-bereiding nog verder geoptimaliseerd kan worden.

CHAPTER

1

General introduction

-1 Introduetion

The Delft Department of Biotechnology (BT) is a world-wide renowned'Wltite Biotechnologu'

center, studying all aspects of industrial fermentation and microbiology. At the onset of the present PhD project, the Analytical Biotechnology Section of BI had just entered a new phase in which proteomics and, in particular, peptidomics techniques were introduced in Delft for the first time, with the aim to develop new methods for innovative approaches in microbiology.

A comprehensive sensitive analysis method for secretory/signaling peptides would not only enable the assay of known ones, but might also open up possibilities to discover novel ones.

Whereas signaling/communication is an essential feature of any living system (celI, tis-sue,organ,organism), it is obvious that not all cells are equally intensely communicative

in terms of peptide signaling, and that specific cell types and/or particular physiological conditions genera te more extensive peptidergic communication than others. This makes it important to select a proper study system ('model') when establishing/optimizing (signa 1-ing) peptide analytics. Only few microbiological peptidergic signaling systems have been described to date, with the yeast mating factor system being the best studied one. A par-allel project to our study,therefore, was initiated to apply newly introduced peptidomics protocols to this model system.The available literature data,including the many data from past physiological experiments as well as the more recent genome sequence annotations do not predict many extra signaling peptides from th is relatively simpIe microbiological sys-tem [13,59, 60). Therefore, it was decided not to rely on the yeast syssys-tem alone to set-up and optimize peptide analytics in Delft. Three other eukaryotic biological systems, known to be highly 'peptide comrnunicative',were taken along in the peptidomics technique intro-duetion phase. These included an easily accessible (insect) neuroendocrine model system (the nervous system being the centraI communication structure within a developed multi-cellular organism), a very rich souree of (amphibian) bioactive peptides as found in exocrine defensive (venom) secretions,and, last but not least, a highly evolved (mammalian) cell type derived from the complex human immune systern, which employs an intricate set of small proteins called cytokines for intercellular crosstalk. The selection of an experimentally ac-cessible hu man immune system model to be translated in an easy cell culture set-up, was one of the challenges taken up in this PhD work.

Additional arguments to select human cells were the completeness and good annotation of the Homo sapiensgenome, as well as the increased interest by Delft University of Tech-nology in Biomedical Research (including'Red BiotecJlIlology'),which had started with the Medical Delta lnitiative and which more recently materialized in the'Delft Health Initiative' 1

This PhD research started of as part of a Marie Curie Research Training Network (MCRTN). This MCRTN, designated "Cellcheck" aimed at the design and implementation of microsystems and microfluidic devices integrating sensors and/or other analysis

Chapter 1

tions for mammalian cell testing 2. The original idea was to develop a microchip-based analysis system which would allow analyses of (the response of) single cells. Our project in the network was originally set out to develop a 'single cell trap' which would eventually enable the analysis of the trapped cell, after being challenged with a variety of extemal stim-uli. This way the device fitted in the original idea of the ABT section to develop methods tostud y cell-to-cell communication. Within Cellcheck devices would be developed for both adhering and suspended cells. At the Analytical Biotechnology group, an earlier project had started within NanoNed netwerk, aiming at the construction of 'yeast cell traps' for the analysis of microbial 'comrnunication' (Herman Walgraeve anofluidics for cell analysis, project number DSF-71353).Ouring the first part of both projects, it became clear that their goals,although technologically very ambitious and challenging, were probably not the most relevant from a biological point of view.This is why, later on in the project, it was decided to abandon the idea of theSingle cell trap,and focus on the miniaturization of cell analysis systems towards the minimal amounts required to allow for biologically relevant (peptide) communicationsigna Is to be analyzedwith the most sensitive ma ss spectrometry systems currently available.

In this chapter we will elaborate on various aspects important to understand the back-ground of the work.

2 Cell-lo-cell Communication

Life of a cell, be it a unicellular microorganism or multicellular organism implies sensing and correctly responding to an always changing microenvironment [76, 99, 137, 146]. An essential feature in the biology of the cell is intercellular comrnunication. Particularly so in rnulticellular organisms,but also in populations of so-called unicellular organisms, intercel-lular comrnunication regulates basic celintercel-lular actlvities through the release of various classes of biomolecules including peptides and (short) proteins. and thereby coordinates cellular behavior for the benefit of the organism (or population) as a whoie.

The human body is one of the more complex multicellular species, consisting of approx-imately 10 trillion cells of more than 200 different types.All these cells live in harmony with each other, under constant threat by extemal factors.In order to counter these threats, good cellular cornmunication is vita!.

Essentially, cell-to-cell communication can occur through physical or chemicaI signa1-ing. Examples of physical signaling include direct connections such as gap-junctions, which comprise intercellular channels interconnecting adjacent cells, allowing rapid exchange of

ions. Another type of close physical contact between cells is the tight junction, which is a site where the membranes of two cells very closely associate, Tight junctions often occur in a belt completely encircling the cello In a sheet of such cells, material is prevenred from passing from one side of the epithelium to the other in between the cells.Instead, the com

-pounds must go through a celI,and hence the passage through the sheet is under cellular

2http: / /www. cel lcheck .eu /

regulation. Two cells can also be connected through tunneling nanotubes. Nanotubes can conneet cells that are several cell diameters apart [7, 18, 24, 46, 62, 107, 174]. Yet another

way of physical signaling is by using electrical signals,changing the membrane potentialof

a cell, or propagation of action potentials [93].

Chemical communication between cells utilizes so-called signaling molecules.

IntercelIu-lar signaling is also known as cell-to-cell communication [7].The extracellular signals can be delivered by different types of messengers amongst which are biogenic amines and amino

acids, steroids, peptides and proteins, and metabolites with conceivable signal functions

such as alkaloids. Stimuli (e.g. horrnones, neurotransmitters or growth factors) acting on cell-surface receptors relay information through intracellular signaling pathways that have

a number of components. They usually begin with the activation of transducers that use

amplifiers to generate internal messengers that either act locally or diffuse throughout the

cell. These messengers then engage sensors that are coupled to the effectors that are

re-sponsible for activating cellular responses [104]. Besides these signaIs, also metabolites can

act as chemical signaling molecules. Many metabolites to which communication functions

can be attributed are found in defensive secretions. Examples of metabolites with signaling

functions can be found in (micro)organisms which exploit these sign aIs to gain

cornpeti-tive advantage. Pathogens can use them to exclude other microbes from a site of infection

[42]. Compounds can be secreted directly into the surrounding medium (by fusing of se-cretory vesicles with the cell membrane; see below), but often also "packed" in so-called

microvesicles or exosomes [18, 122] which bud off from the cell (merocrine secretion). In

special cases, such as in various venom glands, secretion is holocrine, which means that it involves the complete disintegration of the mature gland cell fully packed with secretory material, by which the entire cell content is released into the cell surroundings (usually the outside world).

3 Peptidesand proteins

Peptides and proteins are chains of amino acids connected by peptide bonds in a specific se-quence encoded by the DNA and translated by the ribosome. The shortest peptides are two amino acids long, and with increasing leng th of the amino acid chain, the gene products are designated as"peptides"over"polypeptides"to"proieins'',with a fuzzy border between them. Also the International Union of Pure and Applied Chemistry (IUPAC) does not provide a c1ear weight or amino acid chain leng th limit in its definition of peptides and polypeptides. In literature, peptides are often pragmatically defined, such as "the small proteins running off a typical 20 polyacrylamide gel", or "the small proteins with zero or maximally one tryptic c1eavage site", and,therefore,different upper molecular weight Iimits for peptides can be found ranging from 10 to 30 kDa [85, 89, 90, 124, 175, 176].

Peptide and protein synthesis, processing and secretion are rather complex but very weil regulated mechanisms in the eukaryote cell. The primary structure of secretory proteins (such as signaling peptides) typically beg ins with an aminoterminal signaI peptide sequence

al-Chapter1

ready during synthesis.When amino acid sequence translation by the ribosome is complete, the signal peptide is c1eaved off by a signaI peptidase which releases the mature protein in the ER lumen.So-called transitionary vesicles in which the secretory proteins are packaged, bud off from the ER and travel to the Goigi apparatus, where the primary structure can un-dergo posttranslational modifications. Modifications typical for secreted proteins include glycosylation (addition of sugar moieties/glycans), phosphorylation or sulfatation (addi-tion of phosphate resp. sulfate groups) or intramolecular crosslinks (e.g. disulfide bridges).

Mature proteins destined for secretion finally end up in transport vesicles which ultimately fuse with the cell surface when release is triggered. From that moment secreted proteins become part of the secretome. In this respect, the secretome can be seen as a reflection of thesta te of a cell in a certain environment and, therefore, an important indicator of a cell's condition. thus a souree of potential biomarkers [7, 67, 76, 104, 118).

4 Thesecretome

The secretome of a cell is defined as the complex set of molecules released by living cells to play crucial roles in many physiological and pathological processes [104, 118). It in-c1udes communication signals of various biomolecular classes as outlined above (with

(neuro)hormones and neurotransmitters as the best-known peptide/protein examples), but alsoenzymesand other proteins with an extracellular (supportive) function (such as in con-nective tissuesor biofilms).In multicellular organisrns, secretion can be exocrine when com-pounds are released into the outside world.In so-called endocrine secretion, the compounds are transported to other parts of the organism, e.g.bydiffusing through the circulatory sys-tem (such as the bloodstream). Paracrine secretion is a term used to indicate secretion over a short distance, e.g. to neighboring celis (7).Secretion is not to be confused with excretion, which applies to the discarding of metabolites and other waste products.

5 ThehumanT-cell aspeptide/protein communication model

The immune system is a very complex system involved in many, if not all, diseases and,

therefore, an important part of the human body. ftmakes use of many different cell types, in-tracellular signaling pathways and cell-to-cell communication and relies on the proper func-tioning of white blood cells or leukocytes. By using an intricate set of e.g.cytokines, different types of leukocytes regulate the defense of the body against infectious diseases and foreign 'intruders'. Immune system leukocytes include Iymphocytes, neutrophils, eosinophils, ba-sophiles and monocytes. Of these key players in the immune system, T-Iymphocytes are among the best described. T-cells are distinguishable from other Iymphocytes by the pres-ence of the T-cell receptor (TCR), which becomes activated by peptides presented by the major histocompatibility complex (MHC) (9).

T Iymphocytes expressing the C08 receptor are c1assified as cytotoxic T-Iymphocytes, whereas the expression of the C04 receptor make the cells helper T-Iymphocytes.Helper T-cells (Th) are necessary for handling infections, but they also play a role in auto-immunity.

aïve _{helper T-cells can differentiate into different subtypes of helper 'l-cells, e}

.g.Th l , Th2, depending on the strength and type of antigen and the cytokine environment they are be-ing exposed to (Figure 1.1). Two types of helper T-cells are known for a long time, namely Thl and Th2. Type 1 helper cells drive cell-mediated immune responses with possible tis-sue damage._{Type 2 helper cells are involved in antibody-mediated extracellular responses.} Recently, it became c1ear that more Th subtypes exist, of which Th17 so far is the best char-acterized. Each subset of T-cells has its typical transcription factors by which they can be recognized as weil as a set of cytokines which they secrete. The cytokines produced by these T-cell subsets can negatively influence the function of each other [20, 134, 144, 190,39,57).

SIGNATURE

SIGNATURE PHYSIOLOGICAL

ACTIVATORS _{TRANSCRIPTION}

CYTOKINES FUNCTIONS

FACTORS

Treg _STATSIFOXP3 IL-l0 Tolerance/lmmune

IL-2, TGFIH, TGFI3 suppressien

ATRA

Cellular immunity

~

STAT4/T-bet IFNy _{Clearanceof intracellular}

pathogens

~

Humeral immunity

STAT6/GATA3 IL-4,IL-S, IL-13 Clearance of certain extracellular pathegens Allergy

IL-6, IL-2I,IL-23

Tissue inflammatien STAD IL-17,IL-17F, _Auteimmunity RORytlRORa IL-2I,IL-22 _{Clearance of certain}

extracellular pathegens Figure 1.1: Schematic overview of T-helper cell differentiation, wilh signature transcription factors and cytokines. Functions this specific subset in the human body are indicated.

Activation of helper T-cells typically happens through a 2-step mechanism (Figure 1.2). The first step in this activation is interaction of the TCR with peptides in the MHC c1ass 11 of the antigen presenting cell (APC)._{The second step is the costimulation e.g. the interaction} of CD80 or CD86 receptor on the APC with the CD28 receptor on the helper T-lymphocyte. These steps can be mimicked in vitro_{by either using mitogens Iike phytohaemagglutinin} (PHA), phorbol myristic acetate (PMA)/ionomycin or by using agonistic antibodies against the CD3 and CD28 receptors on the T-cell._{The antibodies can be supplemented in a soluble} form or attached to beads. Upon activation, naïveT-cells are known to be potent inducers of interleukin-Z (IL-2), a 133 amino acid protein which stimulates long-term invitrogrowth of activated T-cell clones [9, 22, 81, 88,103,133,169,217,221).

Throughout the research described in this thesis, both celllines and primary cell cultures have been used. Celllines have the advantage that they are 'imrnortal', being ab Ie to prolif-erate indefinitely, which makes them an attractive system to try-out and perfectionize novel laboratory methods and protocols. A weil described human T-Iymphocyte derived cellline

Chapter 1

isthe Jurkat cell.These cells have been extensivelyused as models for T-cellsignaling and

chemo kine expression, secretion, and activity[1, 77, 80, 116,145,210] .

Primarycell cultures are less artificial cells, as they are harvested straight from their donors, and,therefore, may represent much better real-life aspects within the body. They, however,have a much more limited lifespan, and cannot be obtaine din th eunlimit ed large quantities as the cell lines. After a sma ll number of population doublings, primarycells

undergo senescence andsto p dividing. Further exp erimen ts, therefore,need another clin -ica liso lation. Asdonor material collection requires a clinicalsettin g, their availability for method developmentisverylimited, and, therefore, experiments in primary T-cell cultures areconsiderablymore expensive than incell lines.

This is why, in our first method development experiments, we employed Sup'Tl

and Jurkat -cells. The experime n ts described from cha p te r 4 on, were performed on T-lymphocyte s harvested from theblood of healthydonors,and grown in the laboratory as

so-ca lled primarycultures.

6 Anal ysisof peptides from thesecretome

Asmentionedabove,several different typesof compoundsare actively secreted into the ex-tracellular matrixof acell, amongstwhichare proteinsand peptides,steroid s, aminesand metabo lites. At the Analytic alBiotechnologyGroup focusison the proteins and peptides.

MAPK activation, AP1 formation lonomycin

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APl Nucleus IL-2gen e

Figure 1.2: Simpl ified overviewof T-cellactivation. Dashed lines showche micalactivation, full Iinesarefor the activat ionby an tibodles. Bothwayslead to expre ssionof IL-2 by prior activatienof trans criptionfactorsNFAT and F05-JUN com plex.

However, when examining the proteins and peptides in the extracellular space of cells, not only the actively secreted ones can be found. Several other_{proteins and peptides end up}

in the extracellular medium. A big part of the extracellular space can be filled up with ex-tracellular matrix, a network of macromolecules. _{This matrix is composed of a variety of}

proteins and polysaccharides which are assembied into an organized meshwork in close as-sociation with the surface of the cell that produced them._{Examples of these proteins are (1)}

collagen and elastin, which mainly maintain the structure , and (2) fibronectin and laminin, which have a mainly adhesive function [7). _{Also cellular breakdown proteins may end up}

in the extracellular spa ce as weil as even intracellular proteins after celllysis. All mentioned proteins above are still related to the cell under investigation. In addition, also proteins from extemal origin can end up in the extracellular space. There are the proteins such as serum albumin and other growth factors which are actually necessary in cell cultures, but also proteins that can be unintentionally introduced by the researcher(s), with keratins as the most known ones.It _{is important when examining a cell's secretome to keep all this in}

mind before drawing a conclusion on the proteins and peptides identified in an experiment.

7 _{Modem proteinJpeptide analytics}

As proteomics is for proteins. peptidomics is the comprehensive study of all (native) pep-tides in a biological sample. In the last decade, proteomics has gained increasing recognition as areliabie and reproducible approach to study molecular processes in high-throughput at a global level. Recently also peptidomics is becoming more and more a "hot topic" as it is recognized that peptides play complex regulatory roles in many if not all biological pro-cesses, e.g. intercellular signaling [56, 184). Currently, with a generaI proteomic approach it is possible to detect and identify several hundreds to a few thousands of proteins in a single experiment [38, 100, 136). Central technology in these analyses is mass spectrometry (MS), and more specifically tandem mass spectrometry (MS/MS) [14). With modem mass spec-trometers excelling in sensitivity and dynamic range, also the cell's peptidome has become much more comprehensively accessible for analysis and the discovery of novel biomarkers becomes possible, such as in innovative cancer research [201). The main bottleneck, how-ever, is the large dynamic range in which proteins are present within a biological system.

Human body fluids, especially blood plasma and serum, serve as the most important and readily available sourees for discovering candidate disease biomarkers. However, the de-tection of nov el protein biomarkers, typically present at low concentrations is hampered by the "rnasking" effect caused by a number of highly abundant proteins. Especially the pres-ence of albumin in a sample has been shown to be preventing the successful identification of low abundance biomarkers for many proteomic methods [106, 117, 118, 164, 232). There-fore, it is desirabie to simplify the sample. Different methods for cap turing, partitioning, fractionating, depleting or enriching a sample exist [47, 78).

Ideally, no sample preparation would be required for the arialysis of a sample as every manipulation potentially risks1055_{of sample. In particular for the proteomic/peptidomic} analysis of native peptides, which typically do not require protease (trypsin) digestion prior

Chapter 1

to LC-MS/MS analysis, a so-called "top-down" approachseems logica\. However, asthe complexity of samples still far exceeds the capacity of currently available analyticaI sys-terns, specific sample preparation remains a crucial part of the analysis. Peptidomics sam-ple preparation is often time-consuming and laborious, involving multisam-ple steps [79, 129]. The next chapter presents an overview of different techniques used for the sample prepara-tion and simplificaprepara-tion of complex biologicaI samples for LC-MS/MS based proteinl peptide analysis, including their advantages and possible drawbacks.

8 MassSpeetromet ryand qualitativeproteornies

A mass spectrometer consists of an ion souree. a mass analyzer to separate the rnass-to-charge ratio (mlz)of the ionized analytes (i.c. peptides) and a detector that registers the number of ions at each mlz value. The most commonly used techniques for peptide and protein ionization are matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI). As the latter method allows ionization of peptide analytes directly from so-lution, it is the technique of choice in combination with liquid-based separation methods. In general. mass spectrometer performances are characterized by different parameters. Res-olution is the ability to distinguish between 2 peaks of slightly different mlz ratios. Mass accllracy is the ratio of the mtz measurement error to the true miz and is usually measured in ppm. The dy"amic rallge of an instrument represents the range between the highest signal and the lowest amount of an analyte detected in a single analysis. Very important for MS proteome analyses, i.e. in order to achieve as high a proteome coverage as possi-ble, are furthermore the mass spectrometer'ssensitioituanddllty cycle (sequencing speed) [5,32,35,38,78,86,114,139,141,148,177,178,213,226,228].

The mass analyzer forms the core of the mass spectrometer. Several types exist and var-ious so-called MS/MS (or tandem MS) combinations of analyzers are being used, which is essential for the analysis of complex samples. The mass analyzers used for the research in this thesis comprise a quadrupole time-of-flight arrangement as weil as a combination of a Iinear ion trap and an orbitrap. A Iinear ion trap works with a set of quadrupole rods to con fine ions radially and with a static electrical potentiaIon end electrodes to con fine the ions axially. This Iinear trap can be used as a selective mass filter, or as an actual ion trap MS by creating a potential weil for the ions along the axis of the electrodes. The orbitrap mass analyzer is an electrostatic trap in which the ions oscillate along and around a central spindIe. From the frequency of these oscillations, themfzcan be calculated by Fourier trans-formation. Advantages of the ion trap are high speed and sensitivity, whereas those of the orbitrap are high resolution, high mass accuracy and a dynamic range greater than 103_.

(A) Orbitrap LTQ (B) Orbitrap LTQ

Full MS 30000 UOKI resclutien

11 _~ Ii 11

...

Ii 11

...

iI TIme

...

...

Figure 1.3: Duty cycle Full MS(A) scan versusSIM(6) scan.In Full MSscan mode,ionsare collected in linear ion trap to aset targetvalue after which theyundergoa full MSscan in orbitrap.lons with

intensitiesthat meet therequired target valuearethen fragmented in MS/MS mode. In SIMscan mode, a quick full MSscan isperformed in orbitrap.After this, ions with a m/zinselected rangeare collected in linear ion trap, transferredto orbitrap fora SIMscan and finally, selectedions undergo

fragmentat ionin MS/MSmode.

MS works very weil with peptides as analytes. They areeasilycharged (mostoftenby protonation), and for sizes up to 25-30 amino acids, collision induced dissociation (C ID) allows meaningful (i.e.interpretable) fragmentation(MS/ MS) spectra to be generated.Weil interpretable CID spectra allow full or, at least,partialseq uence annotation of the peptide analyte . Top-down protein mass speetrome try has only very recentlybecome possible, so typicallyproteins are specifically broken down into peptides prior to analysis.The protease trypsin is a verysuitable protease for this. It generates,on average, peptidesof the right length,and, as it c1eavesC-terminally of the basicamino acid residuelysineor arginine, the peptides'ability to be protonatedis assured.

For proteinid en tification, trypticpeptidesare separated byliquid chromatography, on-line fragmented by MS/MSand the data analyzed by database searching.This workflow is alsoknown as shotgun proteomics.A disadvantage of th ismethod for protein identification isthat it dependsgreatly on the qualityof tandem mass spectra. With the current generation of mass spectrometers it is still not possible to get an MS/MS spectru m for every singleion [127]. Thislimitation causes undersampling,and is the reasonwhytechnicaI replicates of the same sample do not exactlyyield identical protein identifications. Reducing the sarn-ple'scomplexitywith one of the techniques described in chapter 2 wiJl reduce the impact ofundersamplingand improve reproducibilityofana lyses. Besides this,also the settings of the massspectrometerhave great influence onthe identificationof proteins.A scan duty cycleof the mass spectrometer is defined asthecombina tion of a MS scan and one or aseries of MS/MSscans. The MS scan can be a full rangeacquisit ionor multiple (overlapping) seg-mented range acquisitions (selected ion monitoring,or SIMscans)(Fig ure 1.3).The number of MS/MSscans is the main determining factor of how long ascan duty cycle take s. To avoid that only highly abundant peptides are selected for fragmentation and MS/MS

ac-Chapter 1

quisition, one can opt for more MS/MS scans per duty cycle. The advantage of the hybrid linear ion trap-orbitrap is that it can perform the MS scan in the orbitrap while at the same

time, MS/MS scans are acquired in the linear iontrap . MS/MS scans aregenerally acquired

in a data-dependent manner (DDA, or data dependent acquisition). This means that only

when a peptide mass reaches a specified intensity threshold, the automatic generation of

fragment ion spectra is triggered for that precursor mass[109, 110, 136, 162, 163].

After acquisition of all MS and MS/MS scans, the data are typ ica lly computerprocessed to create peak lists which are later searched in a database. As for various applications the

currently available computer software does not always suffice, so-calledmalllw/

interpreta-tion of the MS data is often necessary, particularly fordeIWVOsequencing.

A typical (bio)informatics based database search does not interpret the spectrum, but it

annotates the observed fragment ions. Proteins can be identified based on a match of the

mass of tryptic peptides when compared to the insilicodigestion of all proteins. This is

known as peptide mass mapping.When th is search remains inconclusive, or whensamples

are too complex,tandem mass spectra are used to complement these data and to extend the

database search.Wh en searching MS/MSspectra in a database, it is necessary to specify the protease used to reduce search time. However, typically only the minority of the tandem MS spectra allow confident matching with a peptide sequence from the database. This can be due to a variety of reasons such as presence of contaminants and coeluting peptides, bad

qualityspectra, not accounted for post-translational rnodifications, incorrect or imprecise

precursor mass, or missed or exotic c1eavage sites. This lastproblem can of ten be

circum-vented byspecifying "sernitrypsin" as protease when performing a database search.When doingso, the database will be searched for peptides that show tryptic specificity at one

ter-minus, but where the other terminus may be a non-tryptic c1eavage. Itwill only fail to find

peptides that are non-specific at both ends[143, 180, 1921.Proteins can also be falsely

iden-tified.Therefore,the false discovery rate (FDR) is determined.This can be done in different ways, but the most used approach is to re-analyze all data in a so-called "decoy" database.

The FDR is then estimated as the ratio of MS/MS identifications inthe decoy database and

the normal database respectively[163].

Since not only the identification of a protein is of importance but also the differences between different physiological states or time points, it is crucial to have a way to quantify proteins in a sample.Chapter 6 in this thesis discusses some quantitationmethod s and their

advantages and drawbacks.

9 Aimofthe thesi s

Current studies on secreted proteins and peptides typically report results from (targeted) immunological techniques such as enzyme linked immunosorbent assays (ELISA) or from

(indirect) molecular biological techniques such as mRNA expression. These techn iques

however have some drawbacks, including often high costs (appropriate ELISA antibodies are available in limited quantities and are often very expensive), and the fact that they can typically target only one or a few specific proteins or peptides in a single experiment. In

order to monitor several eytokines simultaneously, multiple(x) assays have to be performed [3,22,73,194].

Because MS nowadays enables the identification and quantification of many protein eomponents of complex samples, with theoreticaI attomolar sensitivities, we found it worthwhile to investigate its potential as alternative for the conventional immunoassays [38,100,121, 136].

lnvestigating proteins and peptides secreted by cells is less complex than the analysis of the proteome of a whole eell since it can be performed in eell-free sample preparations. This has the advantage that it avoids purposely induced cell lysis, which provokes unavoid-able sample contamination with non-secreted (e.g., intracellular or membrane) proteins. Nonetheless, secretory protein and peptide analysis is still a big task as (1) their coneentra-tion is typieally only in femtomolar range and (2) the extracellular medium which eellular signa Is are released in typicallyeontains a large complex mixture of background proteins in quantities not seldom present in amounts multiple orders of magnitude higher than the sig-naling peptides,which makes their non-targeted proteomics/peptidomics detection highly ehallenging [29, 104, 118, 119].

The aim of this thesis was to develop and optimize a mass spectrometry based 'p ro-teomics/peptidomics' method for the discovery as weil as analysis of new proteins and peptides secreted by human T-cells.

CHAPTER

2

Sample preparation techniques for the untargeted

LC-MS based discovery of peptides in complex

biological matrices

InezFinoulst, MartijnPinkse, WilliamVan Dongenand PeterVerhaert

This chap ter wasaccept edforpublicat ion

Chapter 2

Abstra ct

Although big progress has been made in sample pretreatment over the last years, there are still considerable limitations when it comes to overcoming complexity and dynamic range problems associated with peptide analyses from biological matrices. Being the little brother of proteomics, peptidemiesis a relatively new field of research aiming at the direct analysis of the smal! proteins. cal!ed peptides, many of which are not amenable for typicaltrypsin based analytics.In this chapter, we present an overview of differenttechniques and methods currently used for reducing a sample's

complexity and for concentrating low abundant compounds to enable successful peptidome anal-ysis.We focus on techniques which can be employed prior to liquid chromatography coupled to mass spectrometry for peptide detection and identification and indicate their advantages as wel! as their shortcomings when it comes to the untargeted analysis of native peptides from complex biological matrices.

1 Introduetion

Peptides are small (1ow molecular weight, LMW) proteins. built up of amino acids con-nected by peptide bonds. _{The shortest peptide is two amino acids long, and with}

increas-ing leng th of the amino acid chain, the name changes from peptideoverpolypeptidetopr o-tein, with a fuzzy border between them._{Also the International Union ofPure and Applied}

Chemistry (IUPAC) has no c1ear weight or amino acid chain length limit. In literature, often pragmatic definitionsare used, such_{as "the small proteins typically running off a typical} 20 polyacrylamide gel", or "the small proteins with zero or maximally one tryptic c1eavage

site", and, therefore, different upper molecular weight limits_{for peptides can be found ra}

ng-ing from 10 to 30 kOa [85,89,90,124,175,176)_{.As proteomics is for proteins. peptidomics is} the comprehensive_{study of all (native) peptides in a biological sample. In the last decade,}

proteomics has gained increasing recognition as areliabie and reproducible approach to study molecular processes in high-throughput ata globallevel. _{Recently also peptidomics}

is becoming more and more a "hot topic" as it is _{recognized that peptides play complex}

regulatory roles in many _{if not all biological processes, e.g. intercellularsignaling [56, 184).}

As such, peptide signals secreted into the extracellular medium, reflect the state of a cell in a certain condition. and, by definition,_{are potential biomarkers indicative for specific p}

hysio-logical/pathological processes [76, 104, 118)._{Currently, with a general proteomie approach}

it is possible to detect and identify several hundreds to a few thousands_{of proteins in a}

sin-gle experiment [38, 100, 1361._{With modern mass spectrometers excelling in sensitivity and}

dynamic range, also the cell's peptidomes become much more comprehensivelyaccessible for analysis and the discovery of novel biomarkers becomes possible,such as in innovative cancer research (201).

Body fluids, especially blood serum or plasma,and,_{in particular cases, (primary) cell}

culture media, serve as typical and readily available sourees for_{a 'peptidornics driven'}

dis-covery of novel candidate disease biomarkers._{However, the detection of peptide}

biomark-ers typically present at low concentrations is hampered by the "rnasking" effect caused by a number of highly abundant proteins [106, 117,118, 164,232). _{The large dynamic}

concen-tration range, in which peptides and proteins_{are present in a biological system presents}

a major bottleneck for peptidomic discovery of new biomarkers. Figure 2.1,reflecting the large dynamic range of proteins _{and peptides in hu man blood plasma, is veryillustrative}

in this respect. _{Especially the presence of albumin in a sample has been shown to} pre-vent the successful identification of low abundant biomarkers_{in many peptidomic studies}

[11,164,198) ._{Hence, different methods for capturing, partitioning, fractionating, depleting}

or enriching a sample have been developed[47,78].

Liquid chromatography (LC) coupled with tandem mass speetrometry (MS/MS) is the analytical method of choice in today's proteomies and peptidomics research.lts major ben

-efits include enhanced specificity (particularly over the GC-MS technologies of 25 years ago, which had very limited applicability for peptide separations), itspotential for high-throughput analyses, no requirement for expensive analyte-specific reagents, high speed of assay development and a relatively low cost per assay (the instrument itself, however, not

Chapter 2

beingthatcheap)(181).

mol/L 10.3

Albumin

IgG

_Classical

10-6

_Plasma

1

Cornplement

Proteins

I

10-9

Insulin

10-12

Tissue leakage

proteins

10-15 10-18 10-24

Figure2.1: IlIustrative iceberg representationof high dynamic range of proteins found in blood,

showing variousclassesof proteinsand peptides (figu re composed of literature data [219] and oth-ers).Four arbitrary assembliesof proteins/peptides can be made and representative species are in-dicated.Icebergtipcontains abundant classicaI plasma proteinsdetectablein 1 ,ti ofsamp le or less.

TIssue leak age proteins typicall yrequ ire,at least,1 mi plasma volumes(typ ically afterdepletionof

interfering abundant proteins),and concentration rangesofsecreted signal peptides /proteinslike ins ulin,somatotropin are yet another3 ordersof magnitude lower.Interleukins and othercytokines really push cu rrent MS systemsto their very limit, whereas other neurosecretory signa I peptides

requ ire extensive concentrationstepsto reachlevelsdetectableby MS.

In an ideal world,no sample preparatien would be required for the analysis of a sample asevery manipulation can lead to problems such as loss of sample. In particular for the proteomic/peptidomicanalysi sof native peptides, which typically do not require protease (trypsin)digestionprior toLC-M5/M5analysis,a so-called'top- d o w n ' approach seems

log-ical. However, as the complexity of samples still far exceeds the capacity of currently avail-able analytical systerns, specific sample preparation remains a crucial part of the analysis in a who Ie. Peptidomics sample preparation is often time-consuming and laborious, involv-ing multiple steps [79, 129]. In this paper we present an overview of different techniques (see Figure 2.2) used for the simplification of complex biological samples and review ad van-tages and possible problems related to them (see Table 2.1). Our focus will be on biofluids such as blood, urine or cerebrospinal fluid (CSF), but some issues related to the peptidome ('secretome') analysis of cultured cells will be considered first.

:

.

Plasma WhoIe blood Urine CSF Saliva Conditioned media etc. Snap freezingl Lyophilization Heat denaturation Protease inhibitor{s} Dye-based depletion Immuno depletion lD-2DPAGE Gel-Free Multidimensional LC-MS Ultrafiltration cv Organic Solvent Extraction

g

SEC

:E RP

êi.

~ lEX

RAM

Figure 2.2: Schematic overview of methods and techniques used in proteome and peptidome analysis for sample preparation prior to LC MS/MS.

2 Sample preparation techniquesfor the analysisof LM W secretome/signaling proteins

2.1 Cellcu lturecond iti ons

Conditioned media are cell culture media which cells have grown in for a certain period of time. The cells "condition" the media by releasing/secreting proteins. cytokines, and other biomolecules. As such, culture supematants or conditioned media (CM) can be considered yet another ('body') fluid that can serve as a souree for the identification of novel biomark-ers, for example in cancer research (154). lt is important to no te that in order to promote a healthy growth of cells in culture, the culture medium has to be supplemented with a stan-dard cocktail of nutrients and growth factors. In mammalian cell cultures this is typically achieved by the addition of a substantial volume of fetal bovine serum (FBS, up to 10% so-lution). When studying theintracellu la r proteome (or peptidome), cells are washed several times with FB5-free cell culture medium and/ or phosphate buffered saline (PBS) prior to lysis to reduce contamination of the sample with (bovine) serum proteins. However, this is not possible when theextra cellular peptidome is under investigation. As a compromise, in most studies the medium containing FBS is replaced by FB5-free medium or medium con-taining reduced amounts of FBSjust before starting the secretome experiment (e.g. giving a biological/physiological stimulus).For adherent cells, it is relatively easy and non-invasive to replace the medium. However, when studying cells in suspension. several centrifugation steps are required before the medium can be replaced, which arguably is a souree of

unnat-Chapter 2

Table 2.1:5ummary of the strengths and weaknesses of analytical tools used in peptidome research as discussed in this chapter

ural stress to the celis, possibly even causing cell lysis. A main disadvantage of working with reduced amounts of FBS (or no FBS at all) is that also this is known to cause metabolic stress to the cells, not seldom inducing cell death and consequently altering the cell culture's secretome [29, 31, 41, 66,118,149,182,227]. Technique Depletion PAGE OFFGel UF Strengths

- Removes highly abundant "household" proteins. allow-ing a 'deeper ' look into the peptdome

- Traditional well-established method

- Able to separate isoforms or PTMs

- Able to remove low molec-ular organic and inorganic irn-purities - Effective pre-fractionation tooi - Medium resolution - Fast - Inexpensive - Easy to automate Weaknesses

- Requires costly antibody columns

- Each protein to be removed requires a different specific an-tibody

- Loss of peptides by non-specific binding

- Unsuitable for highly com-plex samples, poor dynamic range

- Poor resolving power - Proteins/peptides with ex-treme pI values cannot be sep-arated

- The smallest peptides are not retained in MW separation) di-mension

- Very time-consuming and la-borious

- Low dynamic range

- Post concentration is required - Low resolution

- No MWCO discrimination - Long separation times - Variabie quality and repro-ducibility of commercial de-vices

- Loss of hydrophobic peptides by non-specific binding

Table 2.1:Summary of the strengths and weaknesses of analytical tools used in peptidome research as discussed in this chapter

Technique Strengths Weaknesses

- Loading limited by small in-jection volume.

- Complicated LC setup - Extensive application di-rected method development and optimization required. - Extensive method develop-ment for each specific matrix is required

RAM

- High resolution

- Oirectly compatible with MS - High sensitivity in nanoLC - SPE flexible, easy to automate - Efficient pre-concentratien - High resolving power - Reproducible

- Effective removal of HMW compounds

- Relatively large injection vol-umes

- Analysis of untreated sample matrices possible

- Easy to automate

- On-line RAM coupled to (nano-) LC-MS/MS possible

OS - Easy to operate - Tedious to perform

- Inexpensive LC

(SPE/RP/

lEX)

SEC

lEX, ion exchange; LC, liquid chromatography; MWCO, molec-ular weight cut-off; OS, organic solvent extraction; PAGE, poly-acrylamide gel electrophoresis; RAM, restricted access material; RP, reversed phase; SPE, solid phase extraction; UF, u\trafiltra-tion; SEC, size exclusion chromatography

2.2 Gel-e\ectrophoresis (lO-20 PAGE)

A traditionally used and weil established technique in general proteomics is gel elec-trophoresis, mostly in polyacrylamide gels (polyacrylamide gel elecelec-trophoresis, PAGE), fol-lowed by peptide extraction from the gel (when targeting larger (poly)peptides or proteins typically after in-gel digestion) prior to mass spectrometric analysis. With the aid of dena-turing agents, such as SOS, non-covalent intra- and intermolecular protein/protein inter-actions are disrupted by unfolding the macromolecules. Technically it is a simpIe rnethod, and, moreover. it is also robust. However, it has a poor resolving power, which often poses a problem for complex mixtures.Itis often mentioned as an advantage of gel electrophoresis that it increases the depth of proteome/peptidome analysis by fractionating the complex samples and by removing LMW impurities, particularly salts, which may otherwise inter-fere with subsequent MS analyses [10, 55,130,179,212,226,231].

Chapter 2

PAGE can be done in 1 dimension (separating proteins based on their molecular weight), or in 2 dimensions. In 2-dimensional PAGE, the first dimension usually consists of a separa-tion based on the isoelectric point (pI) of proteins/peptides (a so-called isoelectric focusing step), followed by an orthogonal separation according to molecular weight. Two dimen-sional (20) PAGE is very sensitive to molecular charges of a protein (isoelectric focusing) and to the polypeptide length (SOS electrophoresis), making it a very effective method to reveal genetic variants (net charge) and proteolytic c1eavages. This technique however also has some limitations. Proteins/peptides with extreme pI values cannot be separated by this method,and,as indicated above, the smallest peptides are not retained in the second (MW separation) dimension. Another drawback is that 20-PAGE followedbyin-gel diges-tion is very time-consuming and laborious. The dynamic range for detecdiges-tion of 20-PAGE isapproximately102

-104which is less than the protein expression range observed in

bio-logica I systems.These Iimits are illustrated when loading proteins for the detection of low abundant proteins. In order to achieve the detection of low abundant proteins. the protein loading has to be increased. These higher loads,however, typically lead to further reduced resolutiondue to spot fusion and co-migration [11,51,105, 111, 158, 173, 226, 232].

Another important factor in PAGE of proteins is the visualization of the separated pro-teins .Manydifferent protocols exist, each with their advantages and difficulties. The most commonly used visible sta ins are Coomassie brilliant blue (CBB) and silver nitrate staining. CBB staining iseasy to use, compatible with mass spectrometry and Iinear over at least one order of magnitude, 50 it can be used for quantification. The dye binds via electrostatic

interaction with protonated basicamino acids (lysine, arginine and histidine) and by hy-drophobic associations with aromatic residues. Silver nitrate is a more sensitive staining method - 0.5 ng versus 50 ng for CBB - but the staining procedure is more labor intensive and hasa more limited linear range.

Silver staining, though widely regarded as the standard by which all other "ultrasensi-tive" staining methods are judged, remains quite a complex and variabie protein gel sta in-ing methodology, with many dozens of published protocols, all of them requirin-ing several steps. Silver staining may set the standard of rigor for ultimate detection sensitivity but quantitation is not a simpIe matter, due to the complex (polychromatic) nature of the color development step and differences in response factors between different proteins for silver. Other ways to visualize proteins are fluorescent dyes, which are more expensive and require expensive scanners/image analyzers, but also have some advantages. Fluorescent staining methods can combine detection sensitivity that rivals silver staining with workflow advan-tages similar to CBB or zinc ion staining, and offer linear quantitation ranges 1O-100-fold greater than the colorimetrie methods. Oetection is instrumentation dependent. requiring a monochromatic excitation light souree. selective optical filtration to separate the longer waveleng th emitted light from the shorter wavelength (and much brighter) excitation light, and a detection mode [28, 65, 128, 189].

2.3 Off-Gel and Gel-free separation

Because of the limitations associated with gel-based techniques, recently with respect to de-tection of the smaller proteins and peptides (see above), attention has gone to off-gel meth-ods for peptide/protein separations, in particular in solution pi based peptide separations

without the need for carrier ampholytes. Itfocuses proteins and peptides on an

immobi-Iized pH gradient (lPG) gel, which is sealed against a multi-ehamber frame that contains both sample and focusing solutions. The sample is separated by migration through the gel,

followed by diffusion into the weil adjacent to the section of the lPG strip. Itallows for

multiple samples to run simultaneously, requires only small sample volume and no prior sample cleanup. Oisadvantages are that it has a rather long separation time and requires an insulated cooling system [6, 30, 168]. Cologna et al. [30] recently described a varia-tion of the mostly used 'OFFGEL' system (Agilent Technologies. USA). Their device allows customization of the number of wells or the pH gradient. Off-gel separation has not only shown applicability for proteins but also for peptides. Hubner et al. [87] compared the technique to in-gel digestion and found that using peptide off-gel separation led to a third more protein/peptide identifications. Comparing off-gel separation to reversed phase liq-uid chromatography (RPLC) at high pH, Manadas et al. [120] conclude that RPLC leads to the identification of more peptides and also more unique peptides.

In generaI, off-gel separation has clear advantages over a gel-based approach with re-spect to focussing and concentrating peptides, but it still requires further optimization to reach the same level of identifications as an RPLC based separation.

2.4 Specific depletion of highly abundant sample proteins

Many different approaches exist to separate proteins based on their biochemicaI and bio-physical properties such as molecular weight, mass, hydrophobicity. However, these sepa-ration methods are not protein-selective. Another way to reduce a sample's complexity is to specifically remove the most abundant protein(s), by doing (irnmuno-) affinity capturing [4,17,61,64, 149, 164, 167, 198,202,226].

Oepletion of highly abundant proteins can be done based on dyes or on antibodies. An example is the removal of albumin from serum, plasma or cell culture samples. The most used dye for removal of albumin is Cibacron blue (often in combination with protein G for the removal of IgG). This dye however does not only show affinity for albumin but also for NAO, FAO and ATP binding sites of proteins. which often results in the unwanted removal of proteins of interest [53, 236, 239]. Several comparisons have been made on Cibacron blue based depletion of highly abundant proteins from a complex sample versus immunodeple-tion of the same sample. The overall conclusion is that the dye method is less performant than the immuno-based affinity removal: it does not only incompletely bind all albumin from the sample (lower efficacy), but it also appears to remove a substantial portion of pro-teins other than the targeted albumin (lower specificity) [44, 112, 239].

Immunodepletion based on monoclonal antibodies (mAbs) is generally not preferred as, besides being very expensive, these antibodies typically remove only proteins or protein

Chapter 2

fragments with the specific targeted epitope, whereas other fragments of the protein re-main untouched. Therefore, immunodepletion systems are generally based on polyclonal IgG and/or IgY antibodies, targeting multiple epitopes on the same proteins. Moreover, a mixture of polyclonal antibodies to distinct proteins are nowadays commonly used for removing multiple highly abundant proteins at once [239]. Antibody-based depletion of a sample can be performed in (low pressure) spin cartridges or (high pressure) liquid chro-matography (LC) columns.

When using antibodies one has to consider the number of proteins that have to be de-pleted from the sample. Oepending on the system used, it is possible to remove between 1 and 20 abundant proteins. Roche et al. [167] compared several systems which deplete for different amounts of proteins. They observed that increasing the number of depleted pro-teins from 12 to 20 had only little beneficia I effect and could in fact even increase the removal of peptides and proteins of interest which are associated with the abundant proteins. Tu et al. [199] did a comparison of 2 types of multiple affinity removal system (MARS; Agilent Technologies. Inc.) depleting respectively 7 (MARS-7) or 14 (MARS-14) abundant proteins. They also concluded that depleting more proteins is not by definition better. The MARS-14 column removed 7 extra proteins but showed no substantial advantage over the MARS-7 in improving peptide analysis/global protein identifications from plasma.

Recently, a creative way of depleting a sample was developed, the so-called hexapep-tide library of combinatorial pephexapep-tide Iigands. High abundant proteins are expected to quickly saturate their specific affinity Iigands leaving non-bound high abundant proteins to be washed away. In contrast, low and medium abundant proteins and peptides do not saturate their Iigands and hence are concentrated on the beads. This technique has the ad-vantage that peptides and proteins are adsorbed under native conditions and thus allow monitoring of their biological activity [166, 229], although its efficacy is still debated [97].

Limited comparative studies are published on the different depletion and enrichment methods. The few that are, conclude that most of these methods are complementary to each other. Typically the methods compared all lead to identification of a number of peptides and proteins. a part of which is generally identified by all methods under investigation and another part which has been identified uniquely in a sample that was treated with one of the methods [61, 229]. The only exception to this is the comparison of dye-based depletion to immunodepletion of albumin (see above). In this case immunodepletion invariably re-sulted in the most efficient enrichment of low abundant proteins and peptides [44, 112, 239]. Recently, Polaskova et al. [155] compared 6 depletion columns. They, however, did not compare based on identification of proteins but on number of spots found by 20 PAGE and also took into account the costs of all methods. When only looking at protein spots, they concluded that one column (Seppro®MIXE012-LC20, GenWay Biotech) had the best overall performance leading to the largest number of new proteins spots. A second col-umn (Multiple Affinity Removal Colcol-umn human 6, Agilent Technologies) in the same study lead to almost the same quality results, while being cheaper and thence representing a more economical option which could become the preferred method when budgets are limited.