Development of Reagents for Differential Protein Quantitation by Subtractive Parent (Precursor) Ion Scanning

Development of Reagents for Differential Protein Quantitation by

Subtractive Parent (Precursor) Ion Scanning

A° sa Wahlander,† Giorgio Arrigoni,†,‡ Kristofer Warell,† Fredrik Levander,† Ronnie Palmgren,§

Jean-Luc Maloisel,§ Philippe Busson,§ and Peter James*,†

Department of Protein Technology, Lund University, Sweden, Department of Biological Chemistry, Universityof Padova, Italy, and GE Healthcare, Uppsala, Sweden

Received August 24, 2006

We present a generic approach for quantitative differential proteomics that reduces data complexity inproteome analysis by automated selection of peptides for MS/MS analysis according to their isotope-labeling ratio. Isotopic reagents were developed that give products which fragment easily to generatea unique pair of signature ions. Using the ion-pair ratio, we show that it is possible to select only BSApeptides (with a 3:1 light heavy isotope ratio) for MS/MS when spiked in a whole yeast extract usingParent (precursor) Ion Quantitation Scanning (PIQS) for MS/MS.

Keywords: isotope labeling • mass spectrometry • parent-ion • HPLC • reagents • precursor-ion

Introduction

Proteins are responsible for performing and controlling mostof the functions carried out by a cell. Unlike DNA, proteinsshow a massively diverse array of physicochemical propertiesthat makes it unlikely that a generic high-resolution andthroughput separation method will ever be developed. Pres-ently, the most effective method is 2D gel electrophoresis,1,2

although it suffers some limitations.3 Recently, the trend hasbeen toward reducing the physicochemical diversity of proteinsby digestion into peptides at the expense of an increase in themixture complexity. Sample preparation can thereby be simpli-fied and does not have to be optimized for each cell- or tissue-type, as is the case for 2D-PAGE. Peptides behave morepredictably and are more amenable to automated separationby multidimensional chromatography4 and capillary electro-phoresis.5,6 The so-called ‘shot gun’ approach of whole celldigestion has the advantage that all classes of proteins arerepresented, including the traditionally difficult ones, such asmembrane proteins and ones at the extremes of pI and size.

In response to the success of the genome projects, there hasbeen a movement toward the development of method andreagents for isotopic labeling7,8 to enable protein identification,quantification, and the determination of post-translationalmodifications. The first method to be described, IsotopicallyCoded Affinity Tags (ICAT),9,10 used isotopically labeled cys-teine-specific reagents. It provided a means of reducing samplecomplexity by allowing only the Cys-containing peptides to beselectively isolated and quantified. In recent years, many otherisotope labeling approaches have been developed employing

chemical reactions, by enzymatic digestion11 or through meta-bolic incorporation during cell culture.12,13 Quantitative labelingexperiments have been most extensively studied using pairsof isotopes, until recently, when a new technique, employinglabeling with isobaric tags, enabled multiplexed experiments.14

The in vivo strategy has the advantage of enabling labeled andunlabeled samples to be combined earlier in the experimentalprocess than is the case for chemical labeling strategies.

Despite these advances, the main problems remain theextreme dynamic range of protein expression in the cell withup to over 6 orders of magnitude15 and the complexity of thepeptide digest sample, which is compounded by the limitedduty cycle of mass spectrometers currently available. Thisrequires that a reductionist approach be taken, either byenriching for defined subsets of the proteome16 or by usingstrategies for labeling and isolating for certain amino acids aswith the ICAT scheme9 or alternatively by using the elegantCOFRADIC17,18 approach which utilizes diagonal chromatog-raphy of modified peptides for selective proteomics.

We present here a generic approach that allows the selectionof only those peptides from proteins that change in expression,post-translational modification levels for subsequent analysis.We have designed an isotopic label that can be selectivelyattached to peptide N-termini in a digest mixture. The reagentis chemically stable but fragments easily under MS/MS condi-tions to produce a daughter ion of unique mass, common toall labeled peptides, a so-called signature ion. One subset ofproteins is derivatized with the normal “light” form of the label,while another subset is labeled with the deuterated “heavy”form of the same reagent, and two types of signature ions areobtained. The mass spectrometer is operated in parent (pre-cursor) ion scanning mode, alternating scans between light andheavy signature ions. A difference spectrum is generated, andonly those parent peptides showing differences between thetwo labels are selected for MS/MS analysis. The entire workflow

* Corresponding author: Peter James, Protein Technology, BMC D13,Lund University, SE-221 84 Lund, Sweden. E-mail: [email protected]: +46 46 222 1495.

† Lund University.‡ University of Padova.§ GE Healthcare.

10.1021/pr0604312 CCC: $37.00 2007 American Chemical Society Journal of Proteome Research 2007, 6, 1101-1113 1101Published on Web 02/08/2007

is shown schematically in Figure 1. We describe the develop-ment and evaluation of a series of reagents and their perfor-mance in complex mixtures.

Materials and Methods

Materials and Reagents. The brominated synthetic peptidewas a kind gift from Holger Schmidt, ETH Zurich, Switzerland.The corresponding non-brominated peptide was purchasedfrom Cambridge Research Biochemicals Limited (Billingham,U.K.). The acetate salts of (2-mercapto ethyl) trimethyl am-monium, NHS-iodobutyric ester, NHS-iodopropionic ester, andN-nicotinoyloxy succinimide (Nic-NHS-Ester) were from GEHealthcare, Uppsala, Sweden. Albumin (bovine), Glu-fibrin-opeptide, iodoacetic anhydride, 3-amino-1,2,4-triazole-5-thiol,benzene-ethanethiol, 2-diethylamino-ethanethiol HCl, 2-di-methylamino-ethanethiol HCl, 4,6-dimethyl-1,3-dihydro-2H-

benzimidazole-2-thione, 2-mercaptobenzimidazole, 2-mercapto-5-methyl benzimidazole, 2-mercapto-1-methylimidazole, 2-mer-captopyridine, 4-mercaptopyridine, 2-mercapto-4-methylpy-rimidine HCl, Purpald, pyrazine-ethanethiol, 3-iodomethyl-pyridine hydroiodide, sodium hydrosulphide hydrate, andsuccinic anhydride were obtained from Sigma-Aldrich (Stock-holm, Sweden). Mercaptoethylguanidine (MEG) HCl was pur-chased from Calbiochem (EMD Biosciences, Darmstadt, Ger-many), N-guanyl-cys-OH from Bachem (Weil am Rhein,Germany), 4-pyridylethylmercaptane from Toronto ResearchChemicals, Inc. (North York, Canada), and 2-mercaptoben-zimidazole-4,5,6,7-d4 was acquired from QMX LaboratoriesLimited (Essex, U.K.). Sequencing-grade modified trypsin waspurchased from Promega (SDS, Falkenberg, Sweden).

Synthesis of 3-(Thiolmethyl) Pyridine. A total of 900 µL ofsodium hydrosulphide (1 M in H2O) was added to 100 µL of

Figure 1. Schematic drawing of PIQS experimental outline. The two protein samples are first succinylated to prevent the Lys residuesfrom reacting with the N-terminal reagents, followed by enzymatic digestion. The digest mixtures are then treated with iodoaceticanhydride to provide the one-carbon linker and to make the N-termini reactive toward the PIQS label. One digest is labeled with theH4 (light) label and the other with the D4 (heavy) label. The samples are then combined, and excess unreacted reagents are removedusing a HILIC cartridge or a column giving a simultaneous fractionation. The mixture is separated on a RP column before introductionto the mass spectrometer. The mass spectrometer alternates parent ion scanning between the heavy and light signature ions, whilethe PIQS program tracks the appearance of isotopic pairs and determines relative intensity ratios. Only peptides with differential ratiostrigger MS/MS analysis.

research articles WoÄ hlander et al.

1102 Journal of Proteome Research • Vol. 6, No. 3, 2007

Table 1. All of the Reagents Tested in Phase I of the Study Were Used To Derivatize the Synthetic Test Peptide Br-ELYYEa

Parent Ion Quantitation Scanning research articles

Journal of Proteome Research • Vol. 6, No. 3, 2007 1103

3-iodomethyl pyridine hydroiodide (100 mM in H2O). Thereaction was allowed to proceed for 30 min at room temper-ature. The reaction was quenched by lowering the pH to 3 usingconcentrated formic acid. The sample was treated with Tris(carboxyethyl) phosphine (TCEP) (100 mM in H2O) to reduceany disulphide-linked dimers. The product was verified by ESI-MS using a ThermoFinnigan TSQ Quantum (Thermo Electron,Stockholm, Sweden) as described below.

Developing the Reagent: Phase I Chemicals. The chemicalslisted in Table 1 were tested for their suitability to act as asignature ion-generating moiety. Each reagent was reacted withthe peptide BrELYYE. A total of 10 µL of aqueous BrELYYE-peptide solution (1 mM) was diluted to 50 µL with HEPES buffer(100 mM, pH 8), followed by the addition of an equal amountof acetonitrile (ACN). One microliter of N-terminal modifyingreagent (300 mM) was added, and the reaction was allowed toproceed for 30 min in 37 °C before a second addition of anequal amount of N-terminal modifier. After 1 h, the reactionwas quenched by acidification using concentrated formic acid.The peptide samples were concentrated in a Speedvac (ThermoSavant, Techtum Lab AB, Umeå, Sweden), and a small aliquotwas desalted using a ZipTip (Millipore, Stockholm, Sweden).After dilution, the peptides were analyzed by MS and MS/MSon a ThermoFinnigan TSQ Quantum (Thermo Electron, Stock-holm, Sweden) using syringe infusion coupled with a nanosprayionization interface.

Phase II Chemicals. The most effective signature-ion gen-erating reagents from phase I were evaluated further (Table2). The synthetic peptide ELYYE (2 mg/mL in 100 mM, pH 8,HEPES buffer) was diluted to 1 mg/mL with ACN and cooledon ice. The sample was treated with one of the three linkers:iodoacetic anhydride, N-hydroxysuccinimide (NHS)-iodopro-pionic ester, or NHS-iodobutyric ester (200 mM in THF). Thereaction was carried out on ice with three additions of linker

reagent at 3 min intervals. Excess linker was added 200:1 (mol/mol) over peptide. The selected N-terminal modifiers (Table2) were added in three batches in the ratio 100:1 (mol/mol)over peptide, after 0, 10, and 30 min. The reaction was allowedto proceed for 1 h at 37 °C before quenching by lowering thepH of the solution to 3 using concentrated formic acid. ESI-MS and MS/MS analyses of the desalted products wereperformed as described for the phase I samples.

Phase III Testing. The final evaluation of the reagents chosenfrom phase II was carried out using a tryptic digest of bovineserum albumin (BSA) (Table 3). BSA (2 mg/mL in HEPES buffer,200 mM, pH 8) was treated with succinic anhydride, added insmall increments as a solid, to give a final 20-fold molar excessover the calculated amount of Lys residues in protein. The pHwas checked continually and adjusted to 8, and after the finaladdition of anhydride, the samples were incubated for anadditional 15 min at room temperature. Excess unreacted andhydrolyzed succinic anhydride was removed using ProteinDesalting Spin Columns (Pierce, Labdesign Boule Nordic AB,Taby, Sweden) and the buffer exchanged to HEPES buffer (100mM, pH 8) according to the manufacturer’s instructions.Sequencing-grade modified trypsin was added (1:50, w/w) andthe sample incubated for 6 h at 37 °C. The succinylated BSAdigest was diluted 50:50 (v/v) with ACN and the sample puton ice. Peptide N-termini were activated by treatment withiodoacetic anhydride (300 mM in ACN) on ice. The anhydridewas added three times at 3-min intervals to give a total 20-fold molar excess over the estimated trypsin-generated N-termini. The N-terminal modifier (300 mM in ACN or tetrahy-drofuran (THF)) was added at 37 °C at times 0, 10, and 30 min,to a give a total 3-fold molar excess over iodoacetic anhydride.The reaction was allowed to proceed for a further 30 min.Hydroxylamine was added to a final concentration of 6 µM toremove any ester side products (of serine, threonine, or

Table 1 (Continued)

a The table lists the properties under test and gives a short summary of the parameters being evaluated. This test was designed to evaluate the effect of thebasicity of the reagent and the effect of distance of the amine group (linker arm 1) from the sulfur atom bridge on fragmentation.



tyrosine), and the pH adjusted to 11 using NaOH (6 M). After15 min, the reaction was terminated by acidification to pH 2with formic acid.

LC-MS/MS Analysis of Modified BSA. A 6 µL aliquot of themodified BSA peptide mixture (0.16 µg) was injected per runand loaded with a flow rate of 30 µL/min onto a µ-PrecolumnC18 Intersil precolumn (0.3 × 5 mm, 3 µm particle size) from

LC-Packings (Skandinaviska Genetec AB, Gothenburg, Sweden).The sample was separated on an Atlantis dC18 NanoEasecolumn (150 × 0.75 mm, 3 µm particle size) (Waters, Stock-holm, Sweden) at a flow rate of 5 µL/min using a MicromassCapLC unit (Waters, Stockholm, Sweden) and a linear solventgradient (solvent A, H2O, 2% ACN, and 0.1% formic acid; solventB, 10% H2O, 90% ACN, and 0.1% formic acid) from 5% to 80%

Table 2. Reagents Selected from Phase I Were Used To Investigate the Effect of Varying the Distance of the N-Terminal of thePeptide (ELYYE) to the Sulfur Atom Bridge (Linker Arm 2) in Phase II of the Study



B over 45 min. The MS analysis was performed using a Micro-mass Q-TOF Ultima (Waters, Stockholm, Sweden) equippedwith a nanospray source.

Optimization of Reaction Conditions for the BenzimidazoleReagent. A total of 100 µL Glu-fibrinopeptide (1 pmol/µL inH2O) was mixed with 100 µL of HEPES buffer (100 mM, pH 8),

and then further diluted with 200 µL of ACN. Iodoaceticanhydride (300 mM in ACN) was added to the ice-cold solutionin three batches of 4 µL (1:100 of total volume) at 3-min inter-vals. A total of 36 µL of 2-mercaptobenzimidazole (300 mM inTHF) was added at room temperature, in two batches, at times0 and 30 min, to give a final total of 3-fold molar excess relative

Table 3. The Modified Peptides That Were Identified with MASCOT after AutoMS/MS Analysis of the Various Reagent-ModifiedBSA Digests, from Phase III of the Study, Are Listeda



Table 3 (Continued)

a The BSA was succinylated at lysine, and the N-termini of the tryptic peptides were iodoacetylated prior to modification with the reagents listed.



to the iodoacetic anhydride. The reaction was carried out for1 h at 37 °C. An aliquot was desalted using a ZipTip after theiodoacetic anhydride reaction. After the 2-mercaptobenzimi-dazole reaction, aliquots were either washed on ZipTips or usingPolyhydroxyethyl A material (The Nest Group, Inc., Southbor-ough, MA), for hydrophilic clean up in an Eppendorf tube. Thesamples were spotted onto a MALDI target plate and analyzedon a Micromass MALDI LR HT (Waters, Stockholm, Sweden).

Yeast Digestion. Saccharomyces cerevisiae, strain W303-1a(point mutated wild-type with mutations leu2-3/112, ura3-1,trp1-1, his3-11/15, ade2-1, can1-100, GAL SUC2 mal0, genotypeMATa) was grown and harvested at log phase (OD 0.5)according to standard procedures. The yeast was disrupted ina bead beater, and the protein extract was desalted and thebuffer exchanged to HEPES (100 mM, pH 8) using ProteinDesalting Spin Columns (PIERCE, Labdesign Boule Nordic AB,Taby, Sweden). A total of 20 µg of trypsin was added to analiquot of 100 µL (4.966 µg/µL) and the digestion allowed toproceed for 7 h before stopping by acidification and freez-ing.

LC-MS/MS Parent-Ion Quatitation Scanning (PIQS) Analy-sis of Mercaptobenzimidazole-Modified BSA in a Yeast DigestBackground. The BSA sample was split into two aliquots afterthe iodoacetic anhydride reaction. One aliquot was N-termi-nally labeled with 2-mercaptobenzimidazole (H4), and theother with an equal amount of 2-mercaptobenzimidazole-4,5,6,7-d4 (D4) as described above. The samples were combinedin a 60:40 ratio (H4:D4, v/v) prior to separation and analysis.The combined isotopically labeled BSA samples (780 µg) wereloaded in 90% ACN onto a Hydrophilic Interaction LiquidChromatography (HILIC) column (Polyhydroxyethyl A, 200 ×4.6 mm, 5 µm; 300 Å) from PolyLC (The Nest Group, Inc.,Southborough, MA) using a Surveyor autosampler and MS-Pump system from ThermoFinnigan to remove unreactedN-terminal reagent and act as a first-dimension separation. Theelution profile was monitored using a UV-VIS detector (SPD-10A VP/10AVVP) from Shimadzu. Twenty fractions were col-lected at 2 min intervals from 8 to 44 min using a 1 h gradientwith a binary solvent system from 10% to 60% A (A, 15 mMammonium formate, pH 3; B, 100% ACN) at a flow rate of 500µL/min. The collected fractions were dried in a Speedvac(Thermo Savant, Techtum Lab AB, Umeå, Sweden) to removeall organic solvent, and the 8 samples covering the elutionprofile maximum were combined. Approximately, 0.5 µg oflabeled BSA was combined with 10 µg of the tryptic yeastdigestion prior to injection onto a RP-capillary column (Zorbax300SB-C18, 150 × 0.75 mm, 3.5 µm) from Agilent (Stockholm,Sweden), using the 1100 Series capillary and nanopump 2D-

separation system. Peptides were eluted with a 30 min gradientusing a binary solvent system (95:5 f 20:80 (H2O, 0.1% formicacid)/(ACN, 0.1% formic acid)). Analysis was performed usinga ThermoFinnigan TSQ Quantum (ThermoFinnigan, Stock-holm, Sweden) equipped with a nanospray source. The sprayvoltage was set to 1500, parent (precursor) ion scan to 2.5 swith Q1 and Q3 width of 1 and 0.7, and MSMS to 10 s, with Q1and Q3 set to 2 and 0.7, respectively.

LC-MS/MS PIQS Analysis of Both BSA and ADH DigestsModified with Mercaptobenzimidazole in a Yeast DigestBackground. A BSA digest was labeled 60:40 (H4/D4), asdescribed above, and in addition, an approximately equal molaramount of an ADH digest (10-20 pmol), labeled 50:50 (H4/D4), was added. Excess, unreacted reagents were removedusing HILIC cartridges (PolyHydroxyethyl A SPE (Solid-PhaseExtraction) PolyLC, Inc.). The sample was eluted with 20 mMammonium formate, pH 3. As before, a complex backgroundwas provided by adding approximately a 10- to 20-fold molarexcess of tryptic whole cell yeast digest. The mixture (8 µL) wasinjected onto a RP-capillary column (Zorbax 300SB-C18, 150× 0.75 mm, 3.5 µm) from Agilent (Stockholm, Sweden), usingthe 1100 Series capillary and nanopump 2D-separation system.Peptides were eluted at the flow rate 0.3 µL/min with a 30 mingradient using a binary solvent system (95:5 f 20:80 (H2O, 0.1%formic acid)/(ACN, 0.1% formic acid)). Fractions were collectedat 3 min intervals, and analysis was performed using theThermoFinnigan TSQ Quantum (ThermoFinnigan, Stockholm,Sweden) equipped with a nanospray source and PicoTipemitters (New Objective, Woburn, MA). The spray voltage wasset to 1500, parent (precursor) ion scan to 2.5 s with Q1 andQ3 width of 1 and 0.7, and MS/MS to 10 s, with Q1 and Q3 setto 2 and 0.7, respectively.

Data Analysis and Interpretation. The MS and MS/MS datawere analyzed using the MASCOT search engine (Version 2.1.0,Matrix Science, London, U.K.). The database search wasrestricted to bovine tryptic peptides (NCBInr Bos taurus; 7221sequences; 2 354 365 total sequences 2005-03-03 for Q-TOF andNCBInr B. taurus; 39 508 sequences; 3 136 090 total sequences2005-12-24 for TSQ-Quantum) or, for the TSQ-Quantum data,also to tryptic S. cerevisiae peptides (NCBInr baker’s yeast;11 014 sequences; 3 717 264 total sequences 2006-06-18). Forthe Q-TOF data, the precursor error tolerance was set to 0.8Da; for the TSQ-Quantum, it was set to 1.5. All searches wereperformed with 2 missed cleavages allowed, the N-terminalmodification set as variable, and the succinylation as fixed.

Results and Discussion

The basic concept behind PIQS is the differential isotopelabeling of the peptide N-termini after blockage of the ε aminogroup of lysine to prevent these residues from reacting withthe N-terminal label, together with selective parent (precursor)ion scanning.19 Having labeled >99% of all peptides in thecomplex mixture, the reduction in complexity is achieved byselecting only those peptides showing changes in expressionor post-translational modification levels. To achieve this, wedeveloped reagents that modify peptide N-termini, which,although chemically stable, fragment easily under MS/MSconditions to generate a daughter ion at a unique, specific massthat acts as a ‘signature ion’. By switching between parent ionscans for the heavy and light signature ions, we can determinea relative intensity ratio, and it can be used as a criterion fortriggering MS/MS of a particular ion pair, thus, filtering outmost peptides that are not changing (Figure 1).

Figure 2. Schematic structure of the N-terminal of a labeledpeptide. aax stands for the amino acids in the polypeptide chain,and sx is the nomenclature used for the different fragmentationsites under MS/MS conditions. Linker region 1 is between thebasic moiety and the sulfur atom, and linker region 2 is betweenthe N-terminal of the peptide and the sulfur atom.



General Reagent Design. The first objective was to selectthe most suitable label and the best conditions to obtain aspecific and complete reaction with peptide N-termini. Theideal reagent should preferentially fragment at a specific site,generating a strong signature ion that does not coincide in m/zwith any of the commonly occurring immonium and other low-mass ions. Figure 2 shows the nomenclature adopted todescribe the observed fragment ions. A basic charge wasintroduced at the N-terminus to screen for spectra dominatedby b-ions, which is a desirable feature that can facilitate de novosequencing.20

Phase I. In the first stage, reagents with varying degrees ofbasicity were assessed, and the length of the linker region 1,between the amine moiety and the sulfur atom, was varied.The synthetic brominated peptide Br-ELYYE was used toevaluate the phase I compounds for yield of derivatization ofthe peptide N-termini, the generation of a clear signature ion,and the production of strong b-ion series. Compounds withtwo or no carbons in linker region 1, preferentially those witha heterocyclic nitrogen-containing basic moiety (like 2-diethyl-amino ethanethiol, 4-pyridylethylmercaptane, pyrazine-ethane-thiol, 2-mercapto-1-methylimidazole, 3-amino-1, 2, 4-triazole-5-thiole, and 2-mercaptobenzimidazole) were found to give the

best results (Table 1). All of these compounds generated strongsignature ions, sometimes accompanied by a second, weakersignature ion. The spectra were dominated by complete b-ionseries and often accompanied by characteristic a-b ion pairs.Figure 3 shows the MS/MS fragmentation spectrum using2-mercaptobenzimidazole as the N-terminal modifier.

Phase II. In the second phase of the study, the effect ofvarying the chain length between the sulfur atom and theN-terminal of the peptide, linker region 2, was determined. Thereagents from phase I that gave best preliminary results werechosen: 4-pyridylethylmercaptane, 2-mercapto-1-methylimi-dazole, 2-mercaptopyridine, and 4-mercaptopyridine, and thelinkers chosen were one, two, or three carbons in length. Theefficiency of derivatization, strength of the signature ion, andthe b-ion series predominance were again used as the mainevaluation criteria. The results are summarized in Table 2, andan example of an MS/MS spectrum is given in Figure 4. Ingeneral, the results indicated that increasing the linker lengthbeyond a two-carbon spacer decreased the ability to generatea strong signature ion.

Phase III. The final stage of the reagent evaluation wascarried out with the most promising reagents from the secondphase: (2-mercaptoethyl)trimethyl ammonium acetate, 4-py-

Figure 3. MS/MS spectrum of the peptide BrELYYE labeled with 2-mercaptobenzimidazole in the phase I study. This label shows allof the required properties; the spectrum is dominated by a complete b-ion series, with accompanying a-type ions, together with astrong signature ion (m/z 191).



ridylethylmercaptan, 2-mercapto-1-methylimidazole, 3-amino-1,2,4-triazole-5-thiol, 2-mercaptopyridine, 4-mercaptopyridine,and 2-mercaptobenzimidazole. A tryptic digest of succinylatedbovine serum albumin was labeled using iodoacetic anhydridefollowed by the reagent. HPLC-MS/MS analyses of the labeledand unlabeled digests were performed, and the fragmentationspectra were used to search a sequence database with MAS-COT. Table 3 shows the search results and the peptidesidentified. Some of the peptides in Table 3 display unmodifiedterminal lysines. However, these lysine residues are also notmodified by the N-terminal label, which indicates that they arerefractory to modification, either due to structural features orneighboring amino acids. The whole BSA sequence withexpected and actually observed peptides highlighted are pro-vided in Supporting Information.

Two reagents, 2-mercapto-1-methylimidazole and 2-mer-captobenzimidazole, were judged to be the most effective andresulted in the identification of 4 additional peptides identifiedwith concomitant higher total scores, compared to unlabeledBSA. The labeling with these two reagents locates a fixedpositive charge at the N-termini of the peptides that increasesthe intensity of daughter ions in the MS/MS spectra, an effectthat is also seen when the imidazole-like amino acid histidineis at the N-terminus of a peptide.

Final Reagent Selection Criteria. As a result of the screeningprocess, 2-mercaptobenzimidazole in conjunction with io-doacetic anhydride was chosen as the most promising reagent.It gives rise to a strong signature ion at m/z 191 (and 195 forthe D4 deuterated analogue) and marked a-b ions pairs in thefragmentation spectra, with the y-ion series normally absent.The physical properties of the compound are also very favor-

able; it is very soluble in polar organic solvents and does notprecipitate when diluted with aqueous solutions; moreover, ithas a much less offensive odor than some of the otherchemicals and is commercially available in both H4 and D4forms. Another important factor is that the H4 and D4 isoto-pically labeled peptides show almost no differences in retentiontime under standard reverse-phase chromatography conditions.The derivatization of peptides with iodoacetic anhydride and2-mercaptobenzimidazole proceeds rapidly and essentially tocompletion as illustrated in Figure 4 for Glu-fibrinopeptide. Theion at 1553 is the result of cyclization of the N-terminal Glu toform pyroglutamate in the Sigma preparation, which cannotreact with the N-terminal reagents and consequently appearsin all three spectra.

Parent (Precursor) Ion Quantitation Scanning of a Com-plex Mixture. The PIQS approach was first tested using asample of isotopically labeled BSA mixed with a tryptic digestof whole yeast protein extract to provide a complex back-ground. The H4- and D4-labeled BSA samples were combinedin a 60:40 ratio, and after clean up on a HILIC column, thefractions containing the main peptide elution peak werecombined and concentrated. The total ion intensity for theyeast ions was, on average, roughly 30-fold higher for yeast(Figure 5), which is in line with the calculated molar ratio ofabout 20:1, yeast-to-BSA in the sample.

Next, isotopically labeled 60:40 (H4/D4) BSA was mixed with50:50-labeled ADH to make a mixture of labeled peptides, withthe ADH peptide pairs of equal ion intensity, and only the BSApeptide pairs showing a difference in intensity. This was todemonstrate that no peptide pairs would be selected whichfulfilled the isotopic separation criteria only and, furthermore,

Figure 4. The iodoacetic anhydride and the subsequent reaction with 2-mercaptobenzimidazole proceeds essentially to completion onpeptide N-termini. (A) Unlabeled Glu-fibrinopeptide at m/z 1571; (B) Glu-fibrinopeptide labeled with iodoacetic anhydride at m/z 1739;(C) Glu-fibrinopeptide labeled with iodoacetic anhydride and 2-mercaptobenzimidazole at m/z 1761. The ion at 1553 is the result of acontaminant in the peptide standard formed by cyclization of the N-terminal Glu to form pyroglutamate.



that only pairs with a specific difference in intensity ratio wouldbe chosen. In addition, a tryptic digest of whole yeast proteinextract was added to provide complexity. Approximately, equalmolar amounts of labeled BSA and ADH together with roughly20-fold molar excess of yeast were mixed. The samples werecleaned using HILIC cartridges to remove excess and unreactedreagents after labeling.

To select only the BSA-labeled peptides, a program wasdeveloped in ThermoFinnigan Instrument Control Language(ICL) for the Quantum Triple Stage Quadrupole mass spec-trometer. The mass spectrometer is operated in parent (precur-sor) ion scanning mode, taking alternate scans of the parentsof the signature ions at 191 and 195. The program monitorsthe parent ion masses and tracks the appearance of isotopicallylabeled ion pairs. Once a pair of parent ions has been registeredfor five consecutive scans with an intensity ratio fold changeof (1.5 (for 60:40 H4/D4-labeled peptides), the mass spec-trometer switches to MS/MS mode selecting first the H4 thenthe D4 ion and then reverts to parent ion scanning mode. Thecollision energy is set to increase in a linear fashion from alow to a high m/z value during MS/MS, ion pairs selected forfragmentation are stored in memory, and these masses areexcluded from MS/MS for 3 min to avoid data redundancy.The full program is supplied in the Supporting Information.

MS/MS analysis of a labeled tryptic digest of BSA run in theabsence of yeast peptides gave 13 positive peptides identifiedusing the MASCOT search engine compared to only 10 peptideswhen using unlabeled BSA under the same chromatographicconditions (Table 3). This is probably the result of an increasein signal intensity when a basic charge is introduced by thelabel at the N-termini.

The PIQS program selected 16 peptide pairs when analyzingthe sample containing the mix of 60:40 H4/D4-labeled BSA ina 20-fold excess of whole yeast protein tryptic digest (Table 4).Despite the very complex yeast peptide background, 9 of thepairs were identified by both the heavy and the light MS/MSspectra using the MASCOT search engine. Of the 9 pairs, 8 wereidentified as BSA peptides and 1 pair was identified as belong-ing to an unknown B. taurus protein (MGC:127846). Thus, this

Figure 5. Comparison of relative contributions of the BSA peptides (purple) and the yeast tryptic peptides (black) to the total ioncurrent intensity. The abundance of yeast peptides is approximately 30 times higher than those from BSA.

Table 4. BSA Peptide Pairs (60:40 H4/D4) Selected by thePIQS Program and Identified Using the MASCOT SearchEngine from Sample Containing a Complex Yeast Digesta

Nr

H4 parent

(m/z)

D4 parent

(m/z)

H4/D4

ratio (%)

ion

charge sequence

1 315.4 317.3 63/36 2 ?2 428.5 430.5 59/41 2 ?3 632.1 634.2 63/37 2 false pos.4 554.7 556.6 62/38 2 SLGKVGTR5 641.2 643 80/20 2 EKVLTSSAR6 462.5 464.5 61/39 2 VLTSSAR7 368.4 370.4 59/41 2 VASLR8 420.4 422.4 63/37 2 IETMR9 654.6 656.7 70/30 2 false pos.

10 512.4 514.5 63/37 2 false pos.11 517 519 39/61 2 LCVLHEK12 449.5 453.5 61/39 1 *GGAG13 662.7 664.7 59/41 2 ?14 664.7 668.7 58/42 1 ?15 646.8 648.8 65/35 2 ALKAWSVAR16 559.6 561.7 65/35 2 YLYEIAR

a Peptide pairs not identified by MASCOT, but not considered as true fals-positives due to their generation of strong signature ions and well-correlatedspectra, are indicated by a question mark. *Identified as unknown B. taurusprotein (MGC:127846).



appears to be a reasonable number of positive identifications,especially given that not all the fractions from the HILICseparation were combined. The elution profile of the isotopi-cally labeled peptides (Figure 6) shows that the differencebetween light- and heavy-labeled peptides is very small,possibly due to the large π electron cloud of the aromatic ringswhich interact predominantly with the C18 phase and masksthe hydrogen and deuterium atoms at the edge of the ring.

The isotopic ratios calculated by the program agreed wellwith the expected 60:40 (H4/D4) ratio for almost all peptides(Table 4), with only two exceptions. These incorrect ratios werecaused by very early or late sampling in the elution profile ofthe ion pair spectra, where signal intensities are unstable.

The remaining 8 peptide pairs failed to be matched, butupon manual inspection, only four of them could be definedas true “false positives”. These four showed either the presenceof only very weak signature ions and/or pairs of fragmentationspectra that did not correlate with each other. The remainingfour pairs showed spectra that correlated well and showedstrong signature ions; however, the MASCOT searches gave nopositive identifications. Because the PIQS label producesspectra dominated by b-ions rather than the more commony-ion series, this could be a reason for lower recognition andscoring in MASCOT.

From the sample containing both 60:40 (H4/D4) labeled BSAand 50:50 (H4/D4) labeled ADH in the 20-fold excess of yeastdigest, the PIQS program selected 11 pairs of BSA and no ADHpairs (Table 5). In this experiment, a different approach wastaken. The sample mix was subjected to a fast separation on aRP-column with fraction collection. Each fraction was thensubjected to MS analysis using nanospray emitters. Thiseliminates the problem of time-dependent sampling and

resulted in very accurate ratios and no ADH pairs incorrectlyselected. Of the 11 pairs selected, 7 were correctly identifiedwith MASCOT. Upon manual evaluation, two more could beidentified, while the third pair, also unidentified in the previousexperiment, again displayed well-correlating spectra and strongsignature ion signal, but no match could be found in thedatabase.

Conclusions

The PIQS method offers some advantages and complementsthe gel-based approach to proteomics because it is rathernondiscriminatory against type or composition of proteins, and

Figure 6. Elution profile of H4 (green) and D4 (purple) labeled BSA peptides. The peak heights are identical in this case, but the greenscale has been changed to allow the two traces to be easily compared.

Table 5. BSA Peptide Pairs (60:40 H4/D4) Selected by thePIQS Program from Sample also Containing Both 50:50(H4/D4) Labeled ADH and a Tryptic Yeast Digesta

Nr

H4 parent

(m/z)

D4 parent

(m/z)

H4/D4

ratio (%)

ion

charge sequence

1 629.64 633.50 78/22 1 YTR2 554.39 556.34 62/38 2 SLGKVGTR3 368.27 370.27 61/39 2 VASLR4 641.16 643.14 60/40 2 EKVLTSSAR5 420.32 422.24 62/38 2 IETMR6 449.28 453.28 62/38 1 *GGAG7 462.49 464.45 61/39 2 VLTSSAR8 516.64 518.75 61/39 2 LCVLHEK9 559.38 561.24 61/39 2 YLYEIAR

10 646.62 648.63 60/40 2 ALKAWSVAR11 662.25 664.30 61/39 2 ?

a Peptide pairs not identified by MASCOT, but not considered as true false-positives due to their generation of strong signature ions and well-correlatedspectra, are indicated by a question mark.*Identified as unknown B. taurusprotein (MGC:127846).



by varying the proteases used, even membrane proteins areaccessible. Through variation of the prelabeling experiments,the method can be made widely applicable; exchanging thelinker reagent, for example, can enable study of post-transla-tional modifications. Compared to the ICAT reagent, the PIQSlabel benefits both from increasing signal intensity of b-ionsand the almost negligible chromatographic separation betweenthe heavy and light isotope. It is not isobaric like the elegantiTRAQ label and cannot be multiplexed to the same degree; asyet, it does allow dynamic peptide selection in real time.Currently, the reagent is added to the N-terminal in a two-step procedure. However, now that we have selected reagentswith the desired properties, a single stage reagent can besynthesized. The synthesis is simple, and the reagents are verycheap in comparison to those commercially available now.

The usefulness of PIQS, in comparison to other labelingstrategies, lies in the selectivity for peptides with differentialisotope ratios, which, applied to biological samples, will allowthe selection and fragmentation analysis exclusively of peptidesfrom proteins displaying differential expression or post-translational modification. This greatly reduces data complexityand sets it apart from, for example, the iTRAQ approach.14 Thelabel is not restricted to selected amino acids only, as opposedto a number of other isotopic labeling schemes as ICAT9 andhence should give a satisfactory representation of the proteinsin the analyzed sample. When online analysis is used, a fewaberrant ratios can be encountered, but these can be almostcompletely eliminated using prefractionation together withdisposable nanospray emitters such as the Advion system. Theexperiments presented in this paper show how PIQS can beused for targeted studies and works as a proof of principle ofthe method. One application that we are currently investigatingis the use of PIQS for absolute quantitation using ‘proteotypic’or signature peptides selected for proteins of interest in asample. We are also working on the application of the PIQSapproach to whole-proteome studies of biological samples. Theanalysis of such complex samples requires more prefraction-ation prior to the final dynamic analysis, but PIQS is appearingvery useful. Our main current limitation is the lack of verysensitive mass spectrometer with a high duty cycle such as theQ-Trap from Sciex (Toronto, Canada).

Abbreviations: PIQS, parent-ion quantitation scanning.

Acknowledgment. This work was supported by grantsfrom the Knut and Alice Wallenberg Foundation, by a SwegenePostdoctoral program grant to F.L., the Swedish Strategic

Research Council to CREATE Health (P.J.), and from BlanceflorBoncompagni-Ludovisi Stiftelsen to G.A.

Supporting Information Available: The PIQS script,implemented in ICL for the TSQ Quantum (ThermoFinnigan,Stockholm, Sweden) and the sequence of Bovine SerumAlbumin showing the expected and observed peptides high-lighted are available. This material is available free of chargevia the Internet at http://pubs.acs.org.

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Development of Reagents for Differential Protein Quantitation by Subtractive Parent (Precursor) Ion Scanning