C H A

P T E R

15

Sample Depletion,Fractionation, and Enrichment

for Biomarker DiscoveryHaleem J. Issaq, Timothy D. Veenstra

Laboratory of Proteomics and Analytical Technologies, Advanced Technology Program,Frederick National Laboratory for Cancer Research, Frederick, MD, USA

Ph

O U T L I N E

Introduction 23

7

Depletion 238

Fractionation Procedures for Proteinsand Metabolites 238

Affinity Chromatography 241

roteomic and Metabolomic Approaches to Biomarker Discoveryttp://dx.doi.org/10.1016/B978-0-12-394446-7.00015-7 237

Isoelectric Focusing 24

1

Size Exclusion Chromatography 242

Conclusions 243

References 243

INTRODUCTION

The strategy for the discovery of protein ormetabolite biomarkers aims for the identificationof all the proteins and/ormetabolites in a sample.Obviously, complete proteome and metabolomecoverage is presently impossible due to a numberof factors, including their dynamic nature. Thecomplexity of the proteome and metabolomerequires the development of efficient analyticalmethods that involve depletion, fractionation,

concentration, and separation procedures in thehope of detecting the largest possible number ofcompounds. Many attempts have been made inthe last 20 years by analytical chemists to developmethods that possess sufficient resolution to sepa-rate the components of the proteome and metab-olome as well as enough sensitivity to detectcompounds present at the lowest abundances.Because the physicochemical nature of the metab-olome and proteome differ, they require differentfractionation and separation procedures. The

Copyright � 2013 Elsevier Inc. All rights reserved.

15. SAMPLE DEPLETION, FRACTIONATION, AND ENRICHMENT FOR BIOMARKER DISCOVERY238

proteome ismade up of tens of thousands of poly-peptides/proteins that possess the same aminoacid building blocks; however, they vary in sizeand cover a wide range of physical and chemicalproperties (e.g., size, acidity, basicity, hydropho-bicity, hydrophilicity, etc.). Unlike the proteome,the metabolome contains compounds that arestructurallyunrelatedbut alsopossess awide rangeof different chemical and physical properties.Metabolites such as carbohydrates, salts, lipids,steroid hormones, hydrocarbons, and so on oftenrequire different procedures for enrichment, extrac-tion, fractionation, separation, and detection.

Sample fractionation, a procedure for simpli-fying complex mixtures, is an important step inthe search for biomarkers in clinical samples.Fractionationof a sample is different fromaliquot-ing a sample. Aliquoting is a simple procedure inwhich the sample (blood, urine) is divided intosmaller portions (aliquots); fractionation is thedivision of the sample’s “contents” (peptides,proteins, metabolites) into several “groups” (frac-tions) based on their chemical and physical prop-erties, generally using chromatography orelectrophoresis. Extraction, unlike depletion, isa purification step with the aim of selecting andenriching a single or group of compounds ofinterest from a mixture, such as extraction ofestrogen metabolites from urine or serum. Thepurpose of extraction, depletion, and fraction-ation in proteomic and metabolomic analysis isto simplify the complexity of the mixture andallow the detection of low-abundance proteinsand metabolites, resulting in an increase in thenumber of compounds that can be identified.

DEPLETION

Depletion is an essential step in the analysisof the proteome and metabolome. It is a processfor the removal of proteins and other compoundsfrom the sample that can interfere with theanalytical procedure and affect the accuracyof the results or the ability to detect

lower-abundance species. Examples of depletionsteps include the removal of human serumalbumin from blood (serum and plasma) andsalts and lipids from urine. The benefits of deple-tion include: (a) the detection of low-abundanceproteins and low-level metabolites that aremasked by the presence of the depletedcompounds, (b) the disruption of the binding ofmetabolites to proteins, and (c) minimization ofionization suppression when using mass spec-trometry (MS) as a detection device. The proteincontent of blood (serum, plasma) is dominatedby high-abundance proteins (albumin, trans-ferrin, a-1 acid glycoprotein, fibrinogen, cerulo-plasmin, a2-macroglobulin, a1-antitrypsin,apolipoprotein, plasminogen, haptoglobin, andprealbumin) that constitute 99% of the proteincontent of blood (Figure 1). Therefore, only 1%of the entire blood proteome is made up ofproteins that are considered to be in low abun-dance and of particular interest in the search forpotential biomarkers. Therefore, depletion of thehigh-abundance proteins is necessary for thedetection of the low-abundance proteins in blood.The most popular methods for depleting albuminand other high-abundance proteins from bloodinclude immunoaffinity columns or membranefilters.1

Analysis of the metabolome of biologicalsamples such as urine and serum requires theprecipitation of proteins followed by the analysisof the supernatant. Precipitation of proteins canbe achieved by heat or the addition of an acidor organic solvent such as acetonitrile, ethanol,or methanol. Removal of salts, lipids, and so onfrom the sample can be accomplished by columnchromatography, solid phase extraction, orliquideliquid extraction.

FRACTIONATION PROCEDURESFOR PROTEINS AND METABOLITES

Fractionation of protein mixtures prior toseparation and MS analysis can be carried out

22 PROTEINS COMPRISE 99% OF THE PROTEIN MASS IN SERUM!

Fibrinogen

Lipoprotein(a)Factor H

CeruloplasminC4 Complement

Complement Factor BPrealbuminC9 Complement

C1q ComplementC8 Complement

Apolipoprotein A-1

1%

TransferrinlgG Total

lgA TotalAlpha-2-Macroglobulin

lgM TotalAlpha-1-Antitrypsin

C3 ComplementHaptoglobin

10%

Alpha-1-acid Glycoprotein

AlbuminApolipoprotein B

90% 10%

FIGURE 1 Schematic of serum proteome.

FRACTIONATION PROCEDURES FOR PROTEINS AND METABOLITES 239

at the protein or peptide level as indicated inFigure 2. The properties of proteins makethem amenable to different electrophoretic andchromatographic fractionation approaches. Thefirst step in most proteome published studies,after extraction or depletion, is the digestion of

Serum Proteins

Trypsin digestion

SCX-Fractionation

Peptides

X-Fractions

RP-HPLC/MS/MS

Fractionation

X-Fractions

Tryps2D SDS PAGE

RP-H

Protein identification by data base searching

Spots excised

Trypsin digestion

RP-HPLC/MS/MS

SCX

Proteins

the proteins into peptides prior to fractionationby liquid chromatography owing to theirincreased solubility in buffers and solventscommonly used for both reversed-phase andstrong cation exchange (SCX) chromatography(Table 1).

in digestion

PLC/MS/MS

Fractionation

FIGURE 2 Schematic of proteomefractionation procedures at the proteinor peptide level.

TABLE 1 Proteome analysis: intact proteins or proteindigests (peptides)?

Protein mixtures arecomplex

Peptide mixtures are morecomplex

Proteins are less soluble Peptides are more soluble

Proteins are harder tohandle

Peptides are easier to workwith

Loss of one proteinis loss of information

Loss of one peptide is notloss of a protein orinformation

Protein analysis by MSrequires sophisticatedinstruments

Peptide analysis is donewith simpler MSinstrumentation that is moreaccessible

15. SAMPLE DEPLETION, FRACTIONATION, AND ENRICHMENT FOR BIOMARKER DISCOVERY240

Protein and metabolite mixtures are fraction-ated based on their physical and chemical prop-erties by a variety of chromatographic andelectrophoretic methods (Table 2).

TABLE 2 Various proteomic and metabolomicfractionation procedures based onchemical/physical properties

Fractionation Method Property

Ultracentrifugation Density

Size exclusionchromatography (SEX)

Stoke’s radius

Isoelectric focusing (IEF) Isoelectric point

Hydrophobic interactionchromatography (HIC)

Hydrophobicity

Hydrophilic interactionchromatography (HILIC)

Polarity

Reversed-phasechromatography (RP)

Hydrophobicity

Ion exchangechromatography (IEX)

Charge

Affinity chromatography Specific biomolecularinteraction

Gel electrophoresis (GE) Stoke’s radius

Fractionation is achieved by manipulation of(a) the mobile phase properties such as slope(time) of the gradient, organic modifier concen-tration, buffer pH, salt concentration, and/orampholyte pH range; (b) type of column:reversed-phase (e.g., C-18, C-8), ion exchange,size exclusion, normal phase (e.g., silica), oraffinity (use of antibody, lectin, aptamers, metal,DNA, etc.); and (c) gel properties. Therefore,experiments are usually designed to separateproteins and metabolites based on their size,hydrophobicity, polarity, charge, isoelectricpoint, or affinity.

Chromatography, especially HPLC in itsdifferent modes of separation, has been usedextensively for the fractionation of complexmixtures of proteins, peptides, and metabolites.An advantage of HPLC is that it allowsthe selective fractionation of a mixture bymanipulation of the mobile and/or stationaryphases. The principle of chromatographic frac-tionation is based on the interaction of thecompounds of interest with the solid support(stationary phase) and the mobile phase. Theinteraction may be adsorption on silica surfaces,partitioning on reversed phase materials, or ionexchange based on effective charge of theproteins, peptides, and metabolites. Fraction-ation is achieved by using mobile phase gradi-ents, whereby the compounds that areintroduced into the head of the column aredifferentially eluted by changing the organicmodifier concentration with time (RP andHILIC chromatography), the salt content withtime (hydrophobic interaction chromatographyand ion exchange [IEX]), or by mobile phasepH gradient (IEX chromatography). The frac-tionation of peptides has been mostly con-ducted using SCX, a procedure that waspopularized by John Yates and his coworkers2

when they introduced multidimensionalprotein identification technology (MudPIT).The acidified peptide mixture is loaded ontoa biphasic SCX/RP column. Discrete peptidefractions are displaced from the SCX column

ISOELECTRIC FOCUSING 241

directly and are trapped at the beginning RPsection of the biphasic column for separation.This iterative process is repeated multiple timesusing increasing salt gradients. An advantage ofthis separation technology is that the entiresystem is coupled directly online with themass spectrometer enabling a large number ofpeptides to be directly identified in a high-throughput manner. Other chromatographicmodes have been used for peptides, proteins,and metabolites fractionation, as given inTable 2.

Although most fractionation techniques havefocused on the development of methods to frac-tionate the entire proteome samples, methodshave also been developed to interrogate a specificsubset of compounds. For example, Regnier andcoworkers 3 used lectin affinity chromatographyto select glycosylated peptides, significantlyreducing the complexity of the peptide mixtureprior to MS analysis. Dr. Xia Xu et al.4 havedeveloped liquideliquid extraction and MSmethods for analyzing 15 different estrogenmetabolites in biofluid samples such as serumand urine.

AFFINITY CHROMATOGRAPHY

Affinity chromatography is primarily used tocapture a specific protein or a class of proteinswithin complex mixtures. The principle ofaffinity chromatography is based on the abilityof a biologically active molecule to bind specifi-cally and reversibly to a complementary mole-cule, which is often bound to a solid support.These ligand molecules may include antibodies,metals, lectins, biotin, aptamers, and other mole-cules. The binding sites of the immobilizedsubstances must be sterically accessible aftertheir coupling to the solid support and shouldnot be deformed by immobilization. In the caseof specific proteins, an affinant is attached tothe active surface of the column packing materialor column surface. The sample is injected onto

the column and the protein(s) of interest iscaptured by the affinant. Proteins that do notpossess a complementary binding site for thebound ligand will either pass directly throughthe column or be eluted by a low-stringencywashing step. The bound proteins are recoveredby washing the column with a competitivesubstrate or a solution that disrupts the interac-tion between the protein and the affinant (e.g.,denaturants). Although an antibody is directedto a specific protein, many other affinitymethods have been developed to capture a classof proteins. These methods include immobilizedmetal affinity columns containing nickel tocapture histidine-containing peptides,5,6 galliumto isolate phosphopeptides,7 or titanium di-oxides for phosphopeptides.8 In addition,affinity methods have been developed to selectpeptides containing specific types of residuessuch as cysteine, tryptophan, or methionine.9,10

There are a variety of different lectins that havebeen used to selectively separate glycoproteinsbased on the composition of the carbohydrateside-chain.11 A method was also developedthat utilizes phosphoprotein isotope affinitytags, combining stable isotope and biotinlabeling for proteome-wide affinity separationand quantitation of phosphoproteins.12

ISOELECTRIC FOCUSING

The principle of isoelectric focusing (IEF) issimple to understand and perform using eithergel or liquid phase medium. The protein sampleis mixed with the desired pH range carrierampholyte mixture, or other carrier buffer, ina focusing cell or spotted on an SDS-PAGE plate.If an electric potential is applied, the proteinsmigrate to a position in the established pHgradient equivalent to their respective isoelectricpoint (pI). If a protein diffuses away from thispH region, its net charge will change and theresulting electrophoretic forces will influence itsmigration back to its pI point. The net result is

15. SAMPLE DEPLETION, FRACTIONATION, AND ENRICHMENT FOR BIOMARKER DISCOVERY242

the “focusing” of proteins into narrow bands attheir pI values. Liquid-phase IEF allows the frac-tionation of a complexmixture of proteins accord-ing to their pIs in a nongel medium. The fractionscan be collected and further analyzed, if needed,using electrophoresis or chromatography. Thedisadvantages of liquid-phase IEF are that highconcentrations of “neutral” proteins (e.g., whenfocusedat their pI) oftenprecipitate fromsolution.Additionally, the ampholytesused to establish thepH gradient may interfere with subsequent elec-trospray ionization MS analysis. Also, highlyhydrophobic proteinsmay be lost in sample prep-aration or during focusing when the proteinsreach their isoelectric point.13 There are severalIEF devices that can be used to fractionatea complex mixture of proteins at the preparativelevel: (a) Rotofor� cell apparatus, Figure 314; (b)Multicompartment Electrolyzer�with isoelectricmembranes15; (c) recycling IEF (RIEF), pioneeredby Bier et al.16; and (d) free-flow (FF) IEF,17 inwhich the sample medium is recycled througha cooling chamber; in (FF)IEF, samples are contin-uously injected into a carrier ampholyte solution.Complex protein solutions can be easily fraction-ated into a large number of fractions that can becollected and subsequently fractionated using an

FIGURE 3 Rotofor is a liquid-phase isoelectric focusingdevice for the fractionation of a proteome into 20 fractions.

orthogonal chromatographic or electrophoretictechnique.18,19 For comprehensive fractionationand separation, a combination of chromato-graphic and electrophoretic procedures areemployed,20e22 for example, fractionation by gelelectrophoresis followed by digestion of theprotein bands into peptides, which are subse-quently separated using RP chromatography. Itis also possible to fractionate the protein mixtureusing liquid-phase IEF. In this mode, intactproteins separated by IEX are excised from thegel and digested into peptides. These peptidemixtures are then separated by RP-HPLC.Although the combinations of fractionations thatcan be used are almost unlimited, some combina-tions are not particularly efficient, and too manymanipulations can lead to sample loss.

SIZE EXCLUSIONCHROMATOGRAPHY

Size exclusion chromatography (SEC) andelectrophoresis separate proteins according totheir size. Although SEC is an old technique, ithas not been used extensively for proteome frac-tionation due to its comparatively low resolu-tion. In a recent proteomic study by Kosanamet al.,20 SEC was used for the fractionation ofproteins in bronchoalveolar lavage fluid fromlung transplant patients with and withoutchronic graft dysfunction. The proteomes ofbronchoalveolar lavage fluids collected fromfour asymptomatic post-transplant patients andthree patients with symptoms of chronic graftdysfunction were each fractionated into six frac-tions by SEC. The fractions were desalted,concentrated, and analyzed by reversed-phaseHPLC/MS/MS. The study resulted in the identi-fication of 531 proteins. A total of 30 and 39proteins detected exclusively in chronic graftdysfunction and nonchronic graft dysfunction,respectively, were identified as potential candi-dates for the verification phase of this biomarkerdiscovery project. It is not unusual to combine

REFERENCES 243

different extraction procedures in the same studyto achieve the largest number of metabolites/proteins; for example, the pancreatic ascitic fluidproteome was fractionated using a multidimen-sional chromatographic approach that includedsize exclusion, ion exchange, and lectin-affinitychromatography.21 The study resulted in theidentification of 816 proteins, of which 20 over-expressed proteins were identified as putativebiomarker candidates. Also, fractionation ofprotein mixtures may be carried out at theprotein or peptide level using multiple fraction-ation procedures, as mentioned earlier. Metabo-lites are extracted using different solvents thatare compatible with the properties of the metab-olites; different extraction solvents would resultin different metabolic profiles that may affectthe interpretation of the data.22

CONCLUSIONS

The search for protein or metabolite diseasebiomarkers requires the detection and identifi-cation of the largest possible number of thesecompounds in the proteome and metabolomeof clinical samples. To achieve this mission,analytical chemists have devised different chro-matographic and electrophoretic methods forthe depletion, fractionation, and concentrationprior to molecular identification. Depletionand fractionation are essential approaches thatare used to simplify the complexity of the pro-teome and the metabolome and to increase theefficiency of the mass spectrometer. It is notunusual to use a combination of chromato-graphic techniques to achieve comprehensiveproteomic analysis and detection of low-abundance proteins. Although MS and otherinstrumental technologies are often given themost credit for driving proteomics and metabo-lomics discovery, it is a certainty that neither ofthese fields would exist without the invention ofsome of the fractionation techniques discussedin this chapter.

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