Applications of Mass Spectrometry in Proteomics

Izabela Sokolowska,A Armand G. Ngounou Wetie,A

Alisa G. Woods,A,B and Costel C. DarieA,C

ABiochemistry and Proteomics Group, Department of Chemistry and Biomolecular

Science, Clarkson University, 8 Clarkson Avenue, Potsdam, NY 13699-5810, USA.BNeuropsychology Clinic and Psychoeducation Services, SUNY Plattsburgh,

Plattsburgh, NY 12901, USA.CCorresponding author. Email: cdarie@clarkson.edu

Characterisation of proteins and whole proteomes can provide a foundation to our understanding of physiological andpathological states and biological diseases or disorders. Constant development of more reliable and accurate massspectrometry (MS) instruments and techniques has allowed for better identification and quantification of the thousands of

proteins involved in basic physiological processes. Therefore, MS-based proteomics has been widely applied to theanalysis of biological samples and has greatly contributed to our understanding of protein functions, interactions, anddynamics, advancing our knowledge of cellular processes aswell as the physiology and pathology of the human body. Thisreview will discuss current proteomic approaches for protein identification and characterisation, including post-

translational modification (PTM) analysis and quantitative proteomics as well as investigation of protein–proteininteractions (PPIs).

Manuscript received: 27 March 2013.

Manuscript accepted: 14 May 2013.Published online: 3 June 2013.

Introduction

Proteomics is the study of the proteome, which is the compre-hensive complement of proteins in a cell or organism at anygiven time. The proteome of an organism, or even of a single

type of cells, is much more complex than its correspondinggenome. This is primarily due to the changes that can be intro-duced via alternate splicing and post-translational modificationswhich affect virtually all proteins. The proteome differs from

cell to cell and from time to time in its composition depending onthe physiological or pathological state of cells or organisms.Understanding that proteins are the actual effectors of biological

function has been an essential part of biochemistry for over ahundred years.[1] Due to proteomes’ complexity, their analysis isextremely challenging. Therefore, modern biochemical tech-

nologies with improved separation and identification methodshave been introduced.Mass spectrometry (MS) became the coreof advanced methods in proteomics experiments. The integra-tion of MS with a variety of other analytical methods made it

possible to examine virtually all types of samples derived fromtissues, organs, and organisms. MS allowed for the identifica-tion and accurate quantification of thousands of proteins and

peptides from complex biological samples.Of particular interest is the high potential for outcomes from

proteomics experiments to improve the development of diag-

nostics and therapeutic strategies in treating human diseases.[2,3]

Understanding that proteins, their expression level, and inter-actions, influence the physiological function of our body as well

as the development and progression of pathological conditionsis a main force driving current biomedical investigations.Therefore, research aiding protein biomarker discovery is

rapidly growing. Rapid and reliable identification and quantifi-

cation of diagnostic and prognostic markers could improve thequality and length ofmany lives.[4,5] Putative biomarkers are notthe only use of information gained from proteomic studies.

Identification of interactions between proteins and their alter-nations can lead to development of new bio-therapeutics alongwith their targets and potentially to the understanding of theeffects of existing therapies. Although the focus of our review is

on MS and proteomics with biomedical applications, the sameMS and proteomics approaches and techniques also apply tobasic research in other animal systems as well as plants, yeast,

bacteria, viruses, or various pathogens. Therefore, when werefer to biomedical applications, we in fact refer to all organ-isms. This review will provide an overview of current MS for

qualitative identification and characterisation of proteins as wellas its use in quantitative and structural proteomics.

MS Principle and Instrumentation

In proteomic approaches key parameters of MS-based experi-ments are sensitivity, resolution, dynamic range, and mass

accuracy. Therefore, various elements need to be taken intoconsideration during a typicalMS-based proteomics experiment(Fig. 1). Proteomics includes several steps that should be

adapted according to a final experimental goal. First, proteins tobe analysed are isolated from their source and subjected tobiochemical fractionation using techniques such as affinity

purification followed by 1D or 2D gel electrophoresis. Frac-tionated proteins are then subjected to enzymatic digestion(usually with trypsin) to create a mixture of peptides that may or

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http://dx.doi.org/10.1071/CH13137

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Review

RESEARCH FRONT

may not be subjected to additional separation. Peptides are

ionised and introduced into the mass spectrometer where theircharged mass precursor is measured as a mass-to-charge (m/z)ratio and then recorded as a mass spectrum. In MS/MS experi-

ments, the highest intensity precursor ions that meet a set ofuser-defined criteria are selected for collision-induced dissoci-ation (CID) and MS/MS spectra are generated, allowing iden-

tification of peptide sequences.[6–8]

Analysis of peptides and proteins using mass spectrometry

requires their existence as charged ions in the gas phase,allowing their m/z to be measured. To accomplish this, an MSconsists of an ionisation source converting molecules of the

analytes into gas-phase ions, a mass analyser separating ionisedmolecules based on m/z, and a detector recording the number ofions at eachm/z value. TheMS experiments are classified based

on the ion source of the MS and mass analyser type.

Massanalyser

Peptidefragmentation

Massanalyser

Samplefractionation

Proteinseparation

Proteindigestion

Peptideionisation andintroductionto the mass

spectrometer

Peptidemixture

Tandem mass spectrometry (LC-MS/MS)

Protein ID

MS/MSMS

Fig. 1. Schematic of a general proteomic experiment workflow.

Izabela Sokolowska received her M.Sc. in biotechnology at Warsaw University of Life Sciences (2009) and currently she is a

Ph.D. student in the Biochemistry and Proteomics Group at Clarkson University. Her research is focused on the

characterisation of newly discovered protein tumour differentiation factors (TDFs) and mass spectrometry-based approaches

in the analysis of proteins.

Armand G. Ngounou Wetie received his diploma in biotechnology (2008) from the Mannheim University of Applied Sciences,

Germany, and a Master of Science degree in molecular biotechnology from the Technical University of Munich (TUM),

Germany. Currently, Armand is a Ph.D. student in the Department of Chemistry and Biomolecular Science at Clarkson

University (Potsdam, New York), where he is working on understanding the mode of action of the tumour differentiation factor

(TDF) protein.

Dr Alisa Woods is a neurobiologist and mental health counsellor, and currently Research Assistant Professor in the

Department of Chemistry and Biomolecular Science at Clarkson University, as well as Counsellor and Neuropsychology

Researcher at the Neuropsychology Clinic/Psychoeducation Services, SUNYPlattsburgh, New York. The long-term goal of her

research is to understand the connections between biochemistry and behaviour in neurodevelopmental disorders, including

autism and ADHD. She is particularly interested in the identification of protein biomarkers for diagnosis and prognosis of

neurodevelopmental disorders, and in their use for monitoring the psychotherapeutic/behavioural treatment of developmental

disorders.

Dr Costel C. Darie is a biochemist and currently Assistant Professor and leader of the Biochemistry and Proteomics Group

within the Department of Chemistry and Biomolecular Science at Clarkson University. He received his B.S. and M.S. in

biochemistry from Iasi, Romania, and his Ph.D. in biochemistry from Freiburg, Germany. Dr Darie’s main research interests

are in new proteomics approaches for biomarker discovery and identification of post-translational modifications and protein–

protein interactions. His research is also focused on the investigation of one particular protein – the tumour differentiation

factor protein.

722 I. Sokolowska et al.

In most proteomics experiments two types of soft ionisationsare used: matrix assisted laser desorption ionisation (MALDI)[9]

and electrospray ionisation (ESI)[10] (Fig. 2). ESI requires the

analyte to be dissolved in the solvent, ionising peptides andproteins out of solution; therefore it is very often coupled tochromatographic separation tools. Peptides or proteins are

ionised using an electrospray source with high voltage, creatingusually doubly- ormultiply-chargedmolecules. Peptidesmassesare measured based on their precursor ions. In addition, peptide

sequences information can be obtained when precursor ions are

selected for fragmentation, fragmented, and then the fragmentions that correspond to peptides fragment will bemeasured, thusgenerating an MS/MS spectrum. An example of an ESI MS

spectrum of a peptide and its MS/MS fragmentation spectrum ispresented in Fig. 3. ESI ionises molecules of an analyte using apositive or negative ionisation mode. Both large and small

molecules can be either protonated (positive mode) or deproto-nated (negative mode) and generate a distinctive distribution ofcharge states visualised by different MS spectra. Fig. 4 shows

examples of an ESI MS spectrum of intact protein with a

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Mass Spectrometry in Proteomics 723

molecular weight of 16.9 kDa at pH,2 in positive mode ESI(Fig. 4a), and negative mode ESI (Fig. 4b). MALDI ionisesanalytes from a dry, crystalline matrix using a laser beam.

Ionised peptides enter a mass analyser (usually time of flight –TOF) and are further detected as singly-charged molecules. In agenerated MS spectrum, one peak corresponds to one peptideand a list of peaks corresponding to peptides from the same

protein will allow the identification of a protein using peptidemass fingerprinting (PMF) (Fig. 2b). A variation of MALDIionisation is surface-enhanced laser desorption ionisation

(SELDI). It adds an extra purification step by using the bindingof analytes on the probe by ion-exchange, reversed phase,antibody-antigen interactions, and other biochemical affini-

ties.[11–13] This technique uses a high-throughput array-basedapproach with enhanced fast protein profiling, for example, foruse in the discovery of potential diagnostic markers.[14]

Before the development of ESI and MALDI, there wereseveral other hard ionisation methods, such as electron ionisa-tion (EI), chemical ionisation (CI), negative chemical ionisation(NCI), or atmospheric pressure chemical ionisation (APCI).

Hard ionisation results in the breaking of chemical bonds and

the formation of fragment ions.[15,16] Electron ionisation is theoldest mode of ionisation and is still used for organic com-pounds. Using this method, a beam of electrons passes through a

sample in the gas phase and upon collision with a neutralanalyte, produces positively-charged ions (or fragment ions).EI can be used for all volatile compounds (but only smallmolecules with a mass of less than 1000Da) and gives repro-

ducible spectra with structural information derived from frag-mentation but it is not used for molecular weight determinationdue to a poor signal or absence of Mþ ions.[17] Chemical

ionisation is a relatively less energetic mode of ionisation whichgives molecular weight information and reduced fragmentationin comparison to EI, therefore CI can be complementary to EI. In

CI experiments, ionised species are formed when the gaseousmolecules to be analysed collide with primary ions present in thesource under high vacuum.[18] Usually ions formed in MS

experiments are positive but negative chemical ionisation(anions instead of cations formed in the gas-phase) can be alsoused. When using compounds that can produce negative ions,this ionisation technique is very often more sensitive, efficient,

and selective than positive ionisation, but it is also used only for

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(a) Positive ionisation. (b) Negative ionisation. Negative ionisation results in a lower charge distribution due to the loss of

protons from surface-exposed groups in the protein.

724 I. Sokolowska et al.

volatile analytes with amass of less than 1000Da.[19,20] Another

ionisation technique, APCI, uses a reaction between a gas-phaseion and amolecule at atmospheric pressure. A compound unableto be ionised by ESI may be protonated using APCI due to the

presence of stronger gas-phase acids in the source. Often it canbe performed in a modified electrospray source. During APCI,generally only singly-charged ions are formed and it is usuallyapplied to compounds with a molecular weight of less than

1500Da.[21]

There are three main types of mass analyser mass spectro-meters used for proteomics experiments: trapping type

instruments (which include quadrupole ion trap (QIT), linearion trap (LIT), Fourier transform ion cyclotron resonance(FT-ICR), and Orbitrap), quadrupole (Q), and time of flight

(TOF) instruments.Trapping type instruments first accumulate ions in a spe-

cific region and then allow for mass measurement. The iontrap analyser first captures ions in a three-dimensional space

(trap), and then an electrostatic gate pulses to inject ions intothe ion trap. The ion-trap-based analysers are relativelyinexpensive, sensitive, and robust, and therefore a large

amount of proteomic datasets were acquired using ion traps.Because a large number of ions can be collected, this instru-ment typically has high sensitivity. One major problem of the

ion traps is their reduced accuracy in analysing both precursorions in MS mode and fragment ions in MS/MS mode, as wellas reduced accuracy in the analysis of the high molecular mass

of individual proteins or proteomes (top-down proteomics).FT-ICR is also a trapping mass spectrometer, capturing theionised analyte in a high magnetic field under a high vacuum.It is a very sensitive and accurate analyser providing high

resolution, however, transfer of ions into the ICR cell is not avery efficient process, sometimes making these instrumentsnot as sensitive as other mass spectrometers. FT-ICR use is

quite limited in routine proteomics research due to the lowefficiency of peptide fragmentation and the high cost ofinstrumentation.[22,23]

In quadrupole mass analysers, ions are separated based ontheir trajectory in the electric field applied to two pairs ofcharged cylindrical rods. There is an electric potential betweeneach pair of rods drawing the ions towards one rod. In this type of

mass spectrometer, ions are constantly entering the mass ana-lyser. They provide good reproducibility with a relatively smallcost and allow for targeted sample analysis, but their resolution

and accuracy is limited.[16,24]

Instruments with TOF mass analysers are rapidly becomingone of the most popular methods for sample analysis for

proteomics due to their high resolution and relatively low cost.In TOF mass analysers ions are accelerated by a known electricfield. The instrument measures the time it takes ions with

different masses to travel from the ion source to detector,therefore the release of ion needs to be gated. Measurementusing TOF mass analysers are fast and provide high massaccuracy.[16,24]

There can be stand-alone analysers in a single mass spec-trometer, but to take advantage of their strength they are oftenput together in tandem, combining two or three identical or

different analysers (e.g. Q-Trap, QQQ, Q-TOF, TOF-TOF,QQ-LIT). These types of mass spectrometers, referred to ashybrid mass spectrometers, are very powerful and can manipu-

late generated ions to increase sensitivity and resolution, as wellas fragment selected ions for sequence information and modifi-cation identification.[2,24–26]

Protein Identification and Characterisation

Currently there is no method that allows researchers to identifyall proteins of a very complex sample in a one-step experiment.Potentially hundreds to thousands of proteins can be detected

and quantified in complex biological samples. Their relativeabundance often spans 10 or more orders of magnitude, creatingan enormous analytical challenge. To partially solve this prob-

lem, liquid chromatography (LC) separation was coupled withMS. It allows for the analysis of thousands of proteins andpeptides in one measurement.

The very first step in protein identification and characterisa-tion is the determination of its molecular weight (MW) and itsprimary amino acid sequence. There are two major approaches

for protein characterisation using MS: the analysis of intactproteins (top-down approach) and the analysis of a peptidemixture from a digested protein (bottom-up approach), assummarised in Fig. 5. The top-down approach involves the

ionisation of intact proteins in the gas-phase followed by massmeasurement (with high resolution) without prior digestion.Separation and/or fractionation of biological samples into a

single protein or less complex mixtures of proteins are requiredfor accurate measurements. This approach allows for the iden-tification of protein isoforms or any potential post-translational

modifications.[27] In this approach, separated proteins aredigested and directly subjected to peptide mass fingerprintingfor protein identification, or further separated using on-line

tandem mass spectrometry. In the bottom-up variation knownas shotgun proteomics, the protein mixture is directly digestedinto a collection of peptides which are separated using single ormulti-dimensional chromatography and analysed using tandem

MS (MS/MS).[28] An example of LC-MS/MSof a peptide from atandem experiment is shown in Fig. 6. To broaden the charac-terisation of proteins, a combined bottom-up and top-down

approach can be applied.[29,30]

Further complexity in the identification and characterisa-tion of proteins is due to intensive post-translational modifica-

tions (PTMs) of proteins. It is a great challenge to fullyidentify PTM patterns at any given time in cells, tissues, andorganisms and MS-based proteomics became a method ofchoice for their detection and characterisation.[31] PTMs are

important to virtually all biological processes. PTMs are timeand site-specific events that are generally also of low stoichi-ometry, therefore special biochemical enrichment strategies

are used to facilitate their characterisation. Generally, tradi-tional strategies have the capability to characterise most stablemodifications in proteins which includes glycosylation, phos-

phorylation, disulfide bridges, acetylation, ubiquitination, andmethylation. The two most common PTMs in proteins areglycosylation and phosphorylation. General mass spectrometry

approaches for their identification and characterisation aredepicted in Fig. 7.

Glycosylation enhances many biological processes suchas cell-cell recognition and influences a proteins biological

activity.[32,33] In the area of biotheraputics, glycosylation isthe most common modification of recombinant proteins, influ-encing their stability and solubility.[34,35] Identification and

characterisation of glycoproteins is a challenge because glyco-sylation machinery and functionality results in the mixture ofseveral glycosylated variants with different glycan composition.

In MS-based proteomics accurate mass measurements arecritical for accurate calculation of the composition of gly-cans.[36] Selection of the method for fast, accurate, and reliable

Mass Spectrometry in Proteomics 725

identification of glycans is dictated by a research goal. Analysisof glycoproteins may be done using an approach involving

analysis of enzymatically-digested glycopeptides. The resultingmixture is then analysed by LC-MS/MS which allows for theidentification of saccharide diagnostic fragments, particularlyhexoses. This method is not ideal due to poor detection efficiency

for glycoproteins, sometimes poor retention by reversed-phase LC, and their potential for deglycosylation in the gas-phase.[37–39] Therefore, several methods were developed to

address these issues. One strategy involves glycoprotein enrich-ment using lectin affinity chromatography, which facilitatestheir identification in subsequent LC-MS/MS analysis.[40]

Another technique is a chemical method to release glycans fromglycopeptides. N-linked glycans can be released using peptide-N-glycosidase F, while O-linked glycans can be released by

b-elimination. Change in mass units for peptides upon glycanremoval allows establishment of glycosylation sites. ForN-linked glycans, asparagine residues are converted to aspartateleading to an increase of one mass unit. For O-glycans, conver-

sion of threonine residues to aminobutyric acid and serineresidues to alanine, results in loss of 16 mass units.[41] Current

methods allow relatively fast and reliable identification ofglycosylation sites, but accurate characterisation of the exact

structural composition of glycans is still very difficult. Interpre-tation of the spectra obtainedmay be quite challenging due to thepresence of glycan positional isomers and relies on the highpurity of the samples that are analysed, as well as use of

validated standards.[36]

Phosphorylation is a reversible and common PTM that playsa role in controlling and modifying the majority of cellular

processes.[42] Abnormal phosphorylation patterns have beenconnected to various disease states.[43,44] Therefore revealingprotein phosphorylation helps in understanding processes in cell

signalling and may potentially contribute to the discovery ofnew therapeutics and their targets.[45–47] Typically, to detect andcharacterise phosphorylation on peptides, MS/MS fragmenta-

tion in employed. Sometimes, specialised instrument settingsare used, such as scanning of neutral loss in positive ionmode sothat one can detect the loss of a H3PO4 (98 mass units) fromphosphoserine and phosphothreonine residues, and loss ofHPO3

(80 mass units) from phosphotyrosine.[48,49] The ions thatshow these characteristic losses can be further interrogated by

Bottom-up Top-down

Peptide massfingerprinting

Shotgunapproach

Protein mixture

Single protein(or not complex

mixture)

Peptide massmeasurement

Protein ID fromPMF

Peptide mixture

Peptideprecursor massmeasurement

Peptidefragmentation

MS/MS

Protein ID frompeptide

identification

Single protein (orsimple mixture)

Protein precursormass measurement

MSn MS/MS

Protein ID from intactprotein MSn and

MS/MS

Proteinfractionationand separation

MS oronline LC/MS

Targetprecursor ionselection

Digestion

Digestion

Online LC/MS

Fig. 5. Strategies for bottom-up and top-down MS-based protein identification and characterisation.

726 I. Sokolowska et al.

MS/MS analysis. An alternative approach involves complete

methylation of a peptide sample followed by MALDI-basedanalysis in both positive and negative ionisation modes.[50] Asstated, phosphorylation is a transient modification, existing in

nature only at certain time points and conditions, and uponprotein digestion, phosphopeptides are often present in relativelylow abundance relative to non-phosphorylated peptides. Hence

it can be difficult to detect phosphopeptides in traditionalshotgun experiments. Thus, phosphoproteins should beextracted from complex biological mixtures using enrichment

methods. Enrichment of phosphopeptides formass spectrometryanalysis is commonly done using TiO2, metal-oxide basedresins (MOAC), a combination of TiO2 and immobilised metalaffinity chromatography (TiMAC), and antibody affinity

purifications.[51,52]

Another important modification of proteins is the forma-tion of disulfide bridges. They are formed through the oxida-

tion of cysteine residues and are commonly found inextracellular and membrane-bound proteins. The formation

of disulfide bridges plays a significant role in maintaining the

correct function of proteins and as a result the physiologicalstate of an organism. Identification of correct disulfide bridgesis an essential restriction point in solving three-dimensional

structures of proteins.[53,54] Therefore detection of disulfideconnectivities allows researchers to understand the structureof proteins as well as their physiological functions. Investiga-

tion of disulfide-linked proteins can be achieved by separationunder reducing and non-reducing conditions, followed by MSanalysis. The basic MS strategy for disulfide bridge identifi-

cation involves digestion of reduced and non-reduced aliquotsof the same sample and comparing masses of peptides tocharacterise their half-cystinyl residues using MALDI-MS orESI-MS.[55,56]

Many challenges remain in high-throughput large scaleidentification and characterisation of PTMs, but it is obviousthat mass spectrometry-based proteomics has improved our

understanding of those essential components of cellular lifeand can further contribute to this area.

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(b) MS/MS with one double-charged peak at m/z of 809.34 (expanded in the inbox) that corresponds to a peptide with sequence

QVGSGVTTDQVQAEAK which is part of the Ig mu chain C region (gi|7439150). (c) MS/MS fragmentation spectrum produced a

series of y and b ions (marked) that led to identification of a peptide with the amino acid sequence QVGSGVTTDQVQAEAK.

Mass Spectrometry in Proteomics 727

Mass Spectrometry-Based Peptide and ProteinProfiling and Quantitation

Quantitative measurement of changes in protein expressionbetween different physiological and pathological states is

another important area in proteomic research. Several foldchanges in the expression of particular proteins is not uncom-mon, therefore adding a quantitative dimension to mass

spectrometry-based proteomics expands its capabilities andadvantages.[57] Several mass spectrometry-based workflows forprotein and peptide quantification have been developed to assessrelative and absolute amount of protein components in complex

biological samples (Fig. 8). Amain goal of protein profiling andquantification is the development of protein biomarkers, whichis based on the determination of differences in expression levels.

It can be used for diagnosis, disease prognosis, and drugresponse prediction. Therefore, proteomic investigation ofsamples in a clinical setting can enable the high-throughput

identification of protein biomarkers. Traditional gel-basedprotein profiling technologies employ 2D gel methods to com-pare differential expression in two sets of protein mixtures fromdifferent states.[58] Protein spots from 2D gels with different

intensities can be excised and identified using the bottom-upapproach, very often along with their post-translationalmodifications.

MS quantification is usually done using instruments capableof MS/MS fragmentation, especially triple quadrupole or iontrap mass spectrometers. Due to the similar masses of many

compounds in a complex matrix, fragmentation is necessary toaccomplish quantification specificity which can be lacking in asimple MS analysis. The combination of specific precursor ion

and unique fragment ion is used for selective monitoring ofpeptide/protein quantity.[59–61] The quantitative measurementof proteins can be made using label-based or label-free techni-ques. Label-free methods are frequently applied in many

proteomic measurements because of their relative simplicityand low cost.[60] No modification of protein samples are

required (e.g. with stable isotopes), therefore, in theory, anunlimited number of samples can be analysed and the dynamicrange can be quite wide. Two approaches commonly used in

label-free quantification are spectral counting and MS ionintensity (or chromatographic peak area) measurements.[62,63]

In spectral counting, one measures the frequency of selected

peptides associated with a given protein for a MS/MS fragmen-tation.[64,65] The MS ion intensity approach employs high-resolution and high-accuracy LC-MS. Chromatographic peakscorresponding to particular peptides at a normalised elution time

are integrated and used as a quantity measure. The quantity of aprotein or peptide can then be calculated using a standard curveor area under the curve relative to another sample. The dis-

advantage of label-free methods is their heavy dependence onanalytical and biological reproducibility. During analysis anyvariation in sample preparation, ionisation efficiency, or LC-MS

run can lead to increased error in measurements, and as a result,in quantification.[66,67]

To minimise errors from label-free quantification many

current quantification strategies make use of stable isotopes(13C, 15N, 18O, or less desirable 2H).[68] In stable isotopelabelling approaches, native and labelled samples are combinedand analysed simultaneously which reduces errors from tech-

nical variations between measurements. There are countlessreports on the absolute quantification of specific protein usingstable isotope labelled internal standards. The absolute quanti-

fication method relies on the use of synthetic peptide or proteinwith stable isotope labelling on one or more amino acids.[69,70]

They should precisely mimic corresponding endogenous pep-

tides to be used as an internal quantification standard. Labelledpeptides are added directly to the samples to be analysed andduring MS analysis are easily distinguished due to their mass

Biological sample

Glycosylationanalysis

Phosphorylationanalysis

Protein extraction

PNGaseF

Deglycosylatedglycopeptides

Digestion Digestion

Glycopeptideenrichment

Glycans

LC-MS/MS analysis

Peptideidentification

Glycansidentification

Intactglycopeptides

Glycopeptideidentification

Phosphopeptideenrichment(e.g. IMAC)

Phosphopeptides

Phosphopeptidesidentification

�

Fig. 7. General strategies for MS-based characterisation of protein PTMs (glycosylation and phosphorylation).

728 I. Sokolowska et al.

difference. The ratio of signal intensity between native andlabelled peptide accurately indicates the abundance ratio allow-ing for efficient quantification.[71–73] Stable isotope labelling of

select amino acids (usually arginine or lysine) in cell culture(SILAC) is one of the most popular types of metabolic label-ling.[74] It can be used to monitor and quantify in vivo processes

such as protein turnover and to distinguish specific proteininteractions, but it is limited to cell culture and cannot be appliedto other biological matrices.[75] However, recently, Mann’sgroup successfully grew mice on 13Carginine and 13C lysine,

which allows one to compare cells, tissues, bodily fluids, organs,and organisms using SILAC.[76,77] Another commonly usedstrategy for stable isotope labelling is the incorporation of tags

containing stable isotopes into peptides or proteins via chemicalreactions. The first approach using this method, isotope-codedaffinity tag (ICAT), selectively labels sulfhydryl groups on

cysteine residues.[78] To allow simultaneous multiple analysisfrom different biological sources and time-points, chemicallabelling strategies such as isobaric tags for relative and absolute

quantification (iTRAQ) and tandem mass tags (TMT) weredeveloped.[79–81]

Even though those strategies greatly enhanced our quantifi-cation capabilities, they can still suffer from limitations such as alack of reproducibility between individual analysis and false-

negative (missing data) or false-positive identification. Toaddress those issues, targeted quantification approaches suchas selected reaction monitoring (SRM)[82] and multiple reaction

monitoring (MRM)[83] were applied to protein and peptidequantification. These analyses have been performed for manyyears on small molecules and metabolites, but are becomingincreasingly common for quantification of proteins and PTMs.

These experiments are usually performed using a triple quadru-pole tandem mass spectrometer, in which the precursor ion isselected in the first quadrupole, selectively fragmented in the

second collision quadrupole, and fragments are selected in thethird selection quadrupole. This approach predicts fragmentions with good MS response that can be distinguished from

biologicalmatrix interferences. Therefore SRMandMRMshowexcellent reproducibility when used with stable-isotope labelledinternal standards.[84,85]

With current proteomics methodologies and technologies itis inevitable that protein quantification and profiling can

Control

Case

Light isotopelabelling

Heavy isotopelabeling

LC-MS

Case 1

Case 2

Case 3

Isobaric tag 1labelling

Isobaric tag 2labelling

Isobaric tag 3labelling

LC -MS/MS

Precursor ion

MS/MSReporter ions(quantification)

Metabolic labelling – pairwise comparison (e.g. SILAC, ICAT)

Metabolic labelling – multiple comparison (e.g. iTRAQ, TMT)

Chemical labelling – large-scale comparison (e.g. AQUA)

Control

Case 2, 3, 4… n

Proteinextraction

Add internalstandard

Add internalstandard

Proteinextraction

LC-MS

LC-MS

Quantification

Fig. 8. Workflows for MS-based protein quantification strategies using stable isotope labelling.

Mass Spectrometry in Proteomics 729

gradually become a standard diagnostic procedure. Before

large-scale quantitative proteomics involving both label-freeand label-based approaches can be applied in the everyday lifeof clinicians, the reproducibility of large-scale quantification

platforms needs to be developed and maintained over large andprolonged studies. Nevertheless, new emerging technologiesshow promise for disease-specific and/or patient-specific pro-tein-profiling in the near future.

Identification of Protein]Protein Interactions(PPIs) Using MS

All molecular interactions that take place within a cell form theinteractome, and proteins play the main part in this complexnetwork.[86] The interaction of proteins with other biomoleculesgreatly modifies and controls their functions.[87] The distur-

bance of communication between biomolecules would lead tothe malfunctions of cells, tissues, and whole organisms. Manydiseases are associated with one or more defect of PPIs, making

their investigation an essential part of current biomedicalresearch. To increase progress in this area a rather large varietyof methods for identifying PPIs has been developed. Initially,

the stable PPIs or protein complexes were investigated by eithersize exclusion chromatography[88–90] or by sucrose gradientultracentrifugation[91,92]. However, more recently, most of theavailable PPI data (both stable and transient) were discovered by

two high-throughput methods: 1) yeast two-hybrid system(Y2H) and 2) affinity purification MS (AP-MS), allowing fastscreening of a large number of binary interaction (Y2H) and

multiple-protein-complexes (AP-MS).[86,93–97] However, bothY2H and AP-MS have their respective limitations. A largenumber of false positive identifications are observed due to

combining cellular components that would not encounter eachother under physiological conditions.[98–100] To reduce thoseerrors, several modern MS-based methods were applied to

investigate stable and transient PPIs. Native gel electrophoresisseparation techniques allow protein complexes from varioussources to be separated according to their molecular weight andexternal charge, as in blue-native PAGE or based on the internal

charge of subunits in protein complexes as done in colourless-native PAGE.[101,102] Both methods can be further analysedby mass spectrometry to identify components of separated

complexes.[103,104] Additionally, ESI-MS can be used for directmeasurement of stable protein interactions in a solution.A variety of stable protein complexes have been demonstrated to

remain intact in the gas phase during ESI experiments and canbe directly identified in MS without prior separation. Themeasurement of non-denatured native complexes followed bymeasurements of denatured individual components of the same

complex can accurately identify all interactors based onmolecular weight differences.[105,106] As more sensitive andaccurate mass spectrometry-based methodologies are devel-

oped, more components of the complex network of cellularinteractomes will be revealed to serve as a basis for future bio-medical investigations.

Challenges and Perspectives

The biggest challenge for proteomic researchers has been, is,and continues to be, the extreme complexity of proteomes. Our

DNA, with,30000 genes, encodes for,100000 unique proteinsequences.[107,108] LC-MS/MS characterisation of completeproteomes in complex biological samples will require a very

wide dynamic range. The typical dynamic range of detection in

mass spectrometry-based proteomics experiments is 4–6 orders

of magnitude, while in biological samples (such as serum) theprotein concentration can span twice as many orders of mag-nitude. A small number of high-abundance proteins and a large

number of low abundance proteins, which are very often morebiologically relevant during proteomic investigations, makesdetection of most proteins quite difficult. Additional challengessuch as the existence of truncated proteins, splice isoforms,

mutated proteins, as well as the large diversity of potential PTMsmakes the complexity of the samples to be analysed even moredifficult to interpret.[109]

To increase the depth of proteome coverage, most of thecurrent MS-based technologies require a combination of severalsample separation technologies (immunoaffinity, gel electro-

phoresis, multidimensional liquid chromatography etc.) toenhance sensitivity and increase the dynamic range. Anotherproblem associated with MS-based methods is their limitationin high-throughput analysis. We are still years away from a

single-step sample preparation strategy from complex biologi-cal matrixes.

Proteins that are among the most-sought therapeutic targets

include membrane-associated proteins and protein complexes,which make them an important component to be characterisedby MS.[110–113] This subclass of proteome still remains under-

investigated and brings another aspect and challenge to theexperimental approach. The challenges associated with theproteomics of membrane-bound proteins are due to a combina-

tion of nonpolar-transmembrane domains and polar solubledomains. Both soluble and transmembrane domains maycontain PTMs which should be characterised to determine thethree-dimensional structure of proteins. Top-down ESI-MS

technologies have been successfully applied for profiling ofintact proteins, including membrane-associated ones.[114,115]

Recent Advances

Despite the many limitations of MS-based proteomics, we haveto consider its enormous potential and broad range of applica-tions, especially in medicine and diagnostics. It is one of the few

detection methods in which biomarkers from multiple diseasescan be combined into one panel, providingmultiple diagnoses ina single experiment. Great effort must be put into developing

more sensitive, accurate, and reproducible proteomic approa-ches. Stable-labelling of the whole proteome of a given cell,tissue, or organism can be generated. These labelled materialscould potentially be used as universal internal standards in all

biological samples derived from the same matrices.[116,117]

Capabilities of mass spectrometers were greatly enhanced bytheir ion mobility (IM) features which provide information

about the structure of analysed biomolecules. Using this tech-nique, ions that are formed travel through a uniform electricfield at atmospheric pressure, where they become separated.

Time of their travel through the electric field depends on thecross-section of the ions. The larger the ions are, the longer itwill take them to travel to the detector. Ion mobility techniqueshave been an important tool in analytical and physical chemistry

for years, but only recently its potential for the analysis ofproteins has been realised. Measurements taken from ionmobility experiments allow researchers to study the shape and

configuration of proteins and peptides.[118]Many heterogeneousproteins and protein complexes provide a significant challengefor classical structural approaches such as X-ray and NMR.[119]

Therefore, ion mobility LC-IM-MS measurement of proteins

730 I. Sokolowska et al.

and complexes has emerged as an alternative strategy in the field

of structural proteomics, especially due to its potential for thehigh-throughput analysis in four dimensions (mass, elutiontime, drift time, and intensity).[120,121]

Conclusion

In spite of these and other challenges, the impact of MS onclinical and biological research is enormous and is still rapidly

growing. It has substantially increased our understanding ofcomplex biological processes. High-quality reliable MS datacan reveal previously unrecognised cellular connections and

generate new hypotheses regarding signalling pathways, phys-iology, and the development of diseases. However, completecharacterisation of proteomes, both qualitatively and quantita-

tively, is far from being finished. Thanks to the development ofnew MS instrumentation and analysis strategies we are reveal-ing the human proteome one step at a time with the hope that inthe future it will be completely uncovered.

Acknowledgements

We thank Ms. Laura Mulderig, Scott Nichols, and their colleagues (Waters

Corporation) for their generous support in setting up the Proteomics Center

at Clarkson University. CCD thanks Drs. Thomas A. Neubert (New York

University), Belinda Willard (Cleveland Clinic), and Gregory Wolber and

David Mclaughin (Eastman Kodak Co.) for donation of a TofSpec2E

MALDI-MS (each). This work was supported in part by the Keep a Breast

Foundation (KEABF-375–35054), the Redcay Foundation (SUNY

Plattsburgh), the Alexander von Humboldt Foundation, SciFund Challenge,

private donations (Ms. Mary Stewart Joyce &Mr. Kenneth Sandler), and by

the USA Army research office (DURIP grant #W911NF-11–1-0304).

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