Development of Reagents for Differential Protein Quantitation by

Development of Reagents for Differential Protein Quantitation by Subtractive Parent (Precursor) Ion Scanning A° sa Wa˚hlander,† Giorgio Arrigoni,†,‡ Kristofer Wa˚rell,† Fredrik Levander,† Ronnie Palmgren,§ Jean-Luc Maloisel,§ Philippe Busson,§ and Peter James*,† Department of Protein Technology, Lund University, Sweden, Department of Biological Chemistry, University of Padova, Italy, and GE Healthcare, Uppsala, Sweden Received August 24, 2006

We present a generic approach for quantitative differential proteomics that reduces data complexity in proteome analysis by automated selection of peptides for MS/MS analysis according to their isotopelabeling ratio. Isotopic reagents were developed that give products which fragment easily to generate a unique pair of signature ions. Using the ion-pair ratio, we show that it is possible to select only BSA peptides (with a 3:1 light heavy isotope ratio) for MS/MS when spiked in a whole yeast extract using Parent (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 most of the functions carried out by a cell. Unlike DNA, proteins show a massively diverse array of physicochemical properties that makes it unlikely that a generic high-resolution and throughput separation method will ever be developed. Presently, the most effective method is 2D gel electrophoresis,1,2 although it suffers some limitations.3 Recently, the trend has been toward reducing the physicochemical diversity of proteins by digestion into peptides at the expense of an increase in the mixture complexity. Sample preparation can thereby be simplified and does not have to be optimized for each cell- or tissuetype, as is the case for 2D-PAGE. Peptides behave more predictably and are more amenable to automated separation by multidimensional chromatography4 and capillary electrophoresis.5,6 The so-called ‘shot gun’ approach of whole cell digestion has the advantage that all classes of proteins are represented, including the traditionally difficult ones, such as membrane proteins and ones at the extremes of pI and size. In response to the success of the genome projects, there has been a movement toward the development of method and reagents for isotopic labeling7,8 to enable protein identification, quantification, and the determination of post-translational modifications. The first method to be described, Isotopically Coded Affinity Tags (ICAT),9,10 used isotopically labeled cysteine-specific reagents. It provided a means of reducing sample complexity by allowing only the Cys-containing peptides to be selectively isolated and quantified. In recent years, many other isotope labeling approaches have been developed employing * Corresponding author: Peter James, Protein Technology, BMC D13, Lund University, SE-221 84 Lund, Sweden. E-mail: [email protected]. Fax: +46 46 222 1495. † Lund University. ‡ University of Padova. § GE Healthcare. 10.1021/pr0604312 CCC: $37.00

 2007 American Chemical Society

chemical reactions, by enzymatic digestion11 or through metabolic incorporation during cell culture.12,13 Quantitative labeling experiments have been most extensively studied using pairs of isotopes, until recently, when a new technique, employing labeling with isobaric tags, enabled multiplexed experiments.14 The in vivo strategy has the advantage of enabling labeled and unlabeled samples to be combined earlier in the experimental process than is the case for chemical labeling strategies. Despite these advances, the main problems remain the extreme dynamic range of protein expression in the cell with up to over 6 orders of magnitude15 and the complexity of the peptide digest sample, which is compounded by the limited duty cycle of mass spectrometers currently available. This requires that a reductionist approach be taken, either by enriching for defined subsets of the proteome16 or by using strategies for labeling and isolating for certain amino acids as with the ICAT scheme9 or alternatively by using the elegant COFRADIC17,18 approach which utilizes diagonal chromatography of modified peptides for selective proteomics. We present here a generic approach that allows the selection of 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 selectively attached to peptide N-termini in a digest mixture. The reagent is chemically stable but fragments easily under MS/MS conditions to produce a daughter ion of unique mass, common to all labeled peptides, a so-called signature ion. One subset of proteins 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 are obtained. The mass spectrometer is operated in parent (precursor) ion scanning mode, alternating scans between light and heavy signature ions. A difference spectrum is generated, and only those parent peptides showing differences between the two labels are selected for MS/MS analysis. The entire workflow Journal of Proteome Research 2007, 6, 1101-1113

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Figure 1. Schematic drawing of PIQS experimental outline. The two protein samples are first succinylated to prevent the Lys residues from reacting with the N-terminal reagents, followed by enzymatic digestion. The digest mixtures are then treated with iodoacetic anhydride to provide the one-carbon linker and to make the N-termini reactive toward the PIQS label. One digest is labeled with the H4 (light) label and the other with the D4 (heavy) label. The samples are then combined, and excess unreacted reagents are removed using a HILIC cartridge or a column giving a simultaneous fractionation. The mixture is separated on a RP column before introduction to the mass spectrometer. The mass spectrometer alternates parent ion scanning between the heavy and light signature ions, while the PIQS program tracks the appearance of isotopic pairs and determines relative intensity ratios. Only peptides with differential ratios trigger MS/MS analysis.

is shown schematically in Figure 1. We describe the development and evaluation of a series of reagents and their performance in complex mixtures.

Materials and Methods Materials and Reagents. The brominated synthetic peptide was a kind gift from Holger Schmidt, ETH Zurich, Switzerland. The corresponding non-brominated peptide was purchased from Cambridge Research Biochemicals Limited (Billingham, U.K.). The acetate salts of (2-mercapto ethyl) trimethyl ammonium, NHS-iodobutyric ester, NHS-iodopropionic ester, and N-nicotinoyloxy succinimide (Nic-NHS-Ester) were from GE Healthcare, Uppsala, Sweden. Albumin (bovine), Glu-fibrinopeptide, iodoacetic anhydride, 3-amino-1,2,4-triazole-5-thiol, benzene-ethanethiol, 2-diethylamino-ethanethiol HCl, 2-dimethylamino-ethanethiol HCl, 4,6-dimethyl-1,3-dihydro-2H1102

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benzimidazole-2-thione, 2-mercaptobenzimidazole, 2-mercapto5-methyl benzimidazole, 2-mercapto-1-methylimidazole, 2-mercaptopyridine, 4-mercaptopyridine, 2-mercapto-4-methylpyrimidine HCl, Purpald, pyrazine-ethanethiol, 3-iodomethylpyridine hydroiodide, sodium hydrosulphide hydrate, and succinic anhydride were obtained from Sigma-Aldrich (Stockholm, Sweden). Mercaptoethylguanidine (MEG) HCl was purchased from Calbiochem (EMD Biosciences, Darmstadt, Germany), N-guanyl-cys-OH from Bachem (Weil am Rhein, Germany), 4-pyridylethylmercaptane from Toronto Research Chemicals, Inc. (North York, Canada), and 2-mercaptobenzimidazole-4,5,6,7-d4 was acquired from QMX Laboratories Limited (Essex, U.K.). Sequencing-grade modified trypsin was purchased from Promega (SDS, Falkenberg, Sweden). Synthesis of 3-(Thiolmethyl) Pyridine. A total of 900 µL of sodium hydrosulphide (1 M in H2O) was added to 100 µL of

Parent Ion Quantitation Scanning

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

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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 the basicity of the reagent and the effect of distance of the amine group (linker arm 1) from the sulfur atom bridge on fragmentation.

3-iodomethyl pyridine hydroiodide (100 mM in H2O). The reaction was allowed to proceed for 30 min at room temperature. The reaction was quenched by lowering the pH to 3 using concentrated formic acid. The sample was treated with Tris (carboxyethyl) phosphine (TCEP) (100 mM in H2O) to reduce any disulphide-linked dimers. The product was verified by ESIMS using a ThermoFinnigan TSQ Quantum (Thermo Electron, Stockholm, Sweden) as described below. Developing the Reagent: Phase I Chemicals. The chemicals listed in Table 1 were tested for their suitability to act as a signature ion-generating moiety. Each reagent was reacted with the peptide BrELYYE. A total of 10 µL of aqueous BrELYYEpeptide solution (1 mM) was diluted to 50 µL with HEPES buffer (100 mM, pH 8), followed by the addition of an equal amount of acetonitrile (ACN). One microliter of N-terminal modifying reagent (300 mM) was added, and the reaction was allowed to proceed for 30 min in 37 °C before a second addition of an equal amount of N-terminal modifier. After 1 h, the reaction was quenched by acidification using concentrated formic acid. The peptide samples were concentrated in a Speedvac (Thermo Savant, Techtum Lab AB, Umeå, Sweden), and a small aliquot was desalted using a ZipTip (Millipore, Stockholm, Sweden). After dilution, the peptides were analyzed by MS and MS/MS on a ThermoFinnigan TSQ Quantum (Thermo Electron, Stockholm, Sweden) using syringe infusion coupled with a nanospray ionization interface. Phase II Chemicals. The most effective signature-ion generating reagents from phase I were evaluated further (Table 2). The synthetic peptide ELYYE (2 mg/mL in 100 mM, pH 8, HEPES buffer) was diluted to 1 mg/mL with ACN and cooled on ice. The sample was treated with one of the three linkers: iodoacetic anhydride, N-hydroxysuccinimide (NHS)-iodopropionic ester, or NHS-iodobutyric ester (200 mM in THF). The reaction was carried out on ice with three additions of linker 1104


reagent at 3 min intervals. Excess linker was added 200:1 (mol/ mol) over peptide. The selected N-terminal modifiers (Table 2) were added in three batches in the ratio 100:1 (mol/mol) over peptide, after 0, 10, and 30 min. The reaction was allowed to proceed for 1 h at 37 °C before quenching by lowering the pH of the solution to 3 using concentrated formic acid. ESIMS and MS/MS analyses of the desalted products were performed as described for the phase I samples. Phase III Testing. The final evaluation of the reagents chosen from phase II was carried out using a tryptic digest of bovine serum albumin (BSA) (Table 3). BSA (2 mg/mL in HEPES buffer, 200 mM, pH 8) was treated with succinic anhydride, added in small increments as a solid, to give a final 20-fold molar excess over the calculated amount of Lys residues in protein. The pH was checked continually and adjusted to 8, and after the final addition of anhydride, the samples were incubated for an additional 15 min at room temperature. Excess unreacted and hydrolyzed succinic anhydride was removed using Protein Desalting Spin Columns (Pierce, Labdesign Boule Nordic AB, Ta¨by, Sweden) and the buffer exchanged to HEPES buffer (100 mM, pH 8) according to the manufacturer’s instructions. Sequencing-grade modified trypsin was added (1:50, w/w) and the sample incubated for 6 h at 37 °C. The succinylated BSA digest was diluted 50:50 (v/v) with ACN and the sample put on ice. Peptide N-termini were activated by treatment with iodoacetic anhydride (300 mM in ACN) on ice. The anhydride was added three times at 3-min intervals to give a total 20fold molar excess over the estimated trypsin-generated Ntermini. The N-terminal modifier (300 mM in ACN or tetrahydrofuran (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 to remove any ester side products (of serine, threonine, or


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

tyrosine), and the pH adjusted to 11 using NaOH (6 M). After 15 min, the reaction was terminated by acidification to pH 2 with formic acid. LC-MS/MS Analysis of Modified BSA. A 6 µL aliquot of the modified BSA peptide mixture (0.16 µg) was injected per run and loaded with a flow rate of 30 µL/min onto a µ-Precolumn C18 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 NanoEase column (150 × 0.75 mm, 3 µm particle size) (Waters, Stockholm, Sweden) at a flow rate of 5 µL/min using a Micromass CapLC unit (Waters, Stockholm, Sweden) and a linear solvent gradient (solvent A, H2O, 2% ACN, and 0.1% formic acid; solvent B, 10% H2O, 90% ACN, and 0.1% formic acid) from 5% to 80% Journal of Proteome Research • Vol. 6, No. 3, 2007 1105

research articles B over 45 min. The MS analysis was performed using a Micromass Q-TOF Ultima (Waters, Stockholm, Sweden) equipped with a nanospray source. Optimization of Reaction Conditions for the Benzimidazole Reagent. A total of 100 µL Glu-fibrinopeptide (1 pmol/µL in H2O) was mixed with 100 µL of HEPES buffer (100 mM, pH 8),

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and then further diluted with 200 µL of ACN. Iodoacetic anhydride (300 mM in ACN) was added to the ice-cold solution in three batches of 4 µL (1:100 of total volume) at 3-min intervals. A total of 36 µL of 2-mercaptobenzimidazole (300 mM in THF) was added at room temperature, in two batches, at times 0 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-Modified BSA Digests, from Phase III of the Study, Are Listeda

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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.


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

to the iodoacetic anhydride. The reaction was carried out for 1 h at 37 °C. An aliquot was desalted using a ZipTip after the iodoacetic anhydride reaction. After the 2-mercaptobenzimidazole reaction, aliquots were either washed on ZipTips or using Polyhydroxyethyl A material (The Nest Group, Inc., Southborough, MA), for hydrophilic clean up in an Eppendorf tube. The samples were spotted onto a MALDI target plate and analyzed on 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, genotype MATa) was grown and harvested at log phase (OD 0.5) according to standard procedures. The yeast was disrupted in a bead beater, and the protein extract was desalted and the buffer exchanged to HEPES (100 mM, pH 8) using Protein Desalting Spin Columns (PIERCE, Labdesign Boule Nordic AB, Ta¨by, Sweden). A total of 20 µg of trypsin was added to an aliquot of 100 µL (4.966 µg/µL) and the digestion allowed to proceed for 7 h before stopping by acidification and freezing. LC-MS/MS Parent-Ion Quatitation Scanning (PIQS) Analysis of Mercaptobenzimidazole-Modified BSA in a Yeast Digest Background. The BSA sample was split into two aliquots after the iodoacetic anhydride reaction. One aliquot was N-terminally labeled with 2-mercaptobenzimidazole (H4), and the other with an equal amount of 2-mercaptobenzimidazole4,5,6,7-d4 (D4) as described above. The samples were combined in a 60:40 ratio (H4:D4, v/v) prior to separation and analysis. The combined isotopically labeled BSA samples (780 µg) were loaded in 90% ACN onto a Hydrophilic Interaction Liquid Chromatography (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 MSPump system from ThermoFinnigan to remove unreacted N-terminal reagent and act as a first-dimension separation. The elution profile was monitored using a UV-VIS detector (SPD10A VP/10AVVP) from Shimadzu. Twenty fractions were collected at 2 min intervals from 8 to 44 min using a 1 h gradient with a binary solvent system from 10% to 60% A (A, 15 mM ammonium formate, pH 3; B, 100% ACN) at a flow rate of 500 µL/min. The collected fractions were dried in a Speedvac (Thermo Savant, Techtum Lab AB, Umeå, Sweden) to remove all organic solvent, and the 8 samples covering the elution profile maximum were combined. Approximately, 0.5 µg of labeled BSA was combined with 10 µg of the tryptic yeast digestion prior to injection onto a RP-capillary column (Zorbax 300SB-C18, 150 × 0.75 mm, 3.5 µm) from Agilent (Stockholm, Sweden), using the 1100 Series capillary and nanopump 2D1108


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separation system. Peptides were eluted with a 30 min gradient using a binary solvent system (95:5 f 20:80 (H2O, 0.1% formic acid)/(ACN, 0.1% formic acid)). Analysis was performed using a ThermoFinnigan TSQ Quantum (ThermoFinnigan, Stockholm, Sweden) equipped with a nanospray source. The spray voltage was set to 1500, parent (precursor) ion scan to 2.5 s with Q1 and Q3 width of 1 and 0.7, and MSMS to 10 s, with Q1 and Q3 set to 2 and 0.7, respectively. LC-MS/MS PIQS Analysis of Both BSA and ADH Digests Modified with Mercaptobenzimidazole in a Yeast Digest Background. A BSA digest was labeled 60:40 (H4/D4), as described above, and in addition, an approximately equal molar amount of an ADH digest (10-20 pmol), labeled 50:50 (H4/ D4), was added. Excess, unreacted reagents were removed using HILIC cartridges (PolyHydroxyethyl A SPE (Solid-Phase Extraction) PolyLC, Inc.). The sample was eluted with 20 mM ammonium formate, pH 3. As before, a complex background was provided by adding approximately a 10- to 20-fold molar excess of tryptic whole cell yeast digest. The mixture (8 µL) was injected onto a RP-capillary column (Zorbax 300SB-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 at the flow rate 0.3 µL/min with a 30 min gradient using a binary solvent system (95:5 f 20:80 (H2O, 0.1% formic acid)/(ACN, 0.1% formic acid)). Fractions were collected at 3 min intervals, and analysis was performed using the ThermoFinnigan TSQ Quantum (ThermoFinnigan, Stockholm, Sweden) equipped with a nanospray source and PicoTip emitters (New Objective, Woburn, MA). The spray voltage was set to 1500, parent (precursor) ion scan to 2.5 s with Q1 and Q3 width of 1 and 0.7, and MS/MS to 10 s, with Q1 and Q3 set to 2 and 0.7, respectively. Data Analysis and Interpretation. The MS and MS/MS data were analyzed using the MASCOT search engine (Version 2.1.0, Matrix Science, London, U.K.). The database search was restricted to bovine tryptic peptides (NCBInr Bos taurus; 7221 sequences; 2 354 365 total sequences 2005-03-03 for Q-TOF and NCBInr B. taurus; 39 508 sequences; 3 136 090 total sequences 2005-12-24 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). For the Q-TOF data, the precursor error tolerance was set to 0.8 Da; for the TSQ-Quantum, it was set to 1.5. All searches were performed with 2 missed cleavages allowed, the N-terminal modification set as variable, and the succinylation as fixed.

Results and Discussion The basic concept behind PIQS is the differential isotope labeling of the peptide N-termini after blockage of the amino group of lysine to prevent these residues from reacting with the N-terminal label, together with selective parent (precursor) ion scanning.19 Having labeled >99% of all peptides in the complex mixture, the reduction in complexity is achieved by selecting only those peptides showing changes in expression or post-translational modification levels. To achieve this, we developed reagents that modify peptide N-termini, which, although chemically stable, fragment easily under MS/MS conditions to generate a daughter ion at a unique, specific mass that acts as a ‘signature ion’. By switching between parent ion scans for the heavy and light signature ions, we can determine a relative intensity ratio, and it can be used as a criterion for triggering MS/MS of a particular ion pair, thus, filtering out most peptides that are not changing (Figure 1).


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

General Reagent Design. The first objective was to select the most suitable label and the best conditions to obtain a specific and complete reaction with peptide N-termini. The ideal reagent should preferentially fragment at a specific site, generating a strong signature ion that does not coincide in m/z with any of the commonly occurring immonium and other lowmass ions. Figure 2 shows the nomenclature adopted to describe the observed fragment ions. A basic charge was introduced at the N-terminus to screen for spectra dominated by b-ions, which is a desirable feature that can facilitate de novo sequencing.20 Phase I. In the first stage, reagents with varying degrees of basicity 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 to evaluate the phase I compounds for yield of derivatization of the peptide N-termini, the generation of a clear signature ion, and the production of strong b-ion series. Compounds with two or no carbons in linker region 1, preferentially those with a heterocyclic nitrogen-containing basic moiety (like 2-diethylamino ethanethiol, 4-pyridylethylmercaptane, pyrazine-ethanethiol, 2-mercapto-1-methylimidazole, 3-amino-1, 2, 4-triazole5-thiole, and 2-mercaptobenzimidazole) were found to give the

best results (Table 1). All of these compounds generated strong signature ions, sometimes accompanied by a second, weaker signature ion. The spectra were dominated by complete b-ion series and often accompanied by characteristic a-b ion pairs. Figure 3 shows the MS/MS fragmentation spectrum using 2-mercaptobenzimidazole as the N-terminal modifier. Phase II. In the second phase of the study, the effect of varying the chain length between the sulfur atom and the N-terminal of the peptide, linker region 2, was determined. The reagents from phase I that gave best preliminary results were chosen: 4-pyridylethylmercaptane, 2-mercapto-1-methylimidazole, 2-mercaptopyridine, and 4-mercaptopyridine, and the linkers chosen were one, two, or three carbons in length. The efficiency of derivatization, strength of the signature ion, and the b-ion series predominance were again used as the main evaluation criteria. The results are summarized in Table 2, and an example of an MS/MS spectrum is given in Figure 4. In general, the results indicated that increasing the linker length beyond a two-carbon spacer decreased the ability to generate a strong signature ion. Phase III. The final stage of the reagent evaluation was carried out with the most promising reagents from the second phase: (2-mercaptoethyl)trimethyl ammonium acetate, 4-pyJournal of Proteome Research • Vol. 6, No. 3, 2007 1109

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Figure 4. The iodoacetic anhydride and the subsequent reaction with 2-mercaptobenzimidazole proceeds essentially to completion on peptide 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 a contaminant in the peptide standard formed by cyclization of the N-terminal Glu to form pyroglutamate.

ridylethylmercaptan, 2-mercapto-1-methylimidazole, 3-amino1,2,4-triazole-5-thiol, 2-mercaptopyridine, 4-mercaptopyridine, and 2-mercaptobenzimidazole. A tryptic digest of succinylated bovine serum albumin was labeled using iodoacetic anhydride followed by the reagent. HPLC-MS/MS analyses of the labeled and unlabeled digests were performed, and the fragmentation spectra were used to search a sequence database with MASCOT. Table 3 shows the search results and the peptides identified. Some of the peptides in Table 3 display unmodified terminal lysines. However, these lysine residues are also not modified by the N-terminal label, which indicates that they are refractory to modification, either due to structural features or neighboring amino acids. The whole BSA sequence with expected and actually observed peptides highlighted are provided in Supporting Information. Two reagents, 2-mercapto-1-methylimidazole and 2-mercaptobenzimidazole, were judged to be the most effective and resulted in the identification of 4 additional peptides identified with concomitant higher total scores, compared to unlabeled BSA. The labeling with these two reagents locates a fixed positive charge at the N-termini of the peptides that increases the intensity of daughter ions in the MS/MS spectra, an effect that is also seen when the imidazole-like amino acid histidine is at the N-terminus of a peptide. Final Reagent Selection Criteria. As a result of the screening process, 2-mercaptobenzimidazole in conjunction with iodoacetic anhydride was chosen as the most promising reagent. It gives rise to a strong signature ion at m/z 191 (and 195 for the D4 deuterated analogue) and marked a-b ions pairs in the fragmentation spectra, with the y-ion series normally absent. The physical properties of the compound are also very favor1110


able; it is very soluble in polar organic solvents and does not precipitate when diluted with aqueous solutions; moreover, it has a much less offensive odor than some of the other chemicals and is commercially available in both H4 and D4 forms. Another important factor is that the H4 and D4 isotopically labeled peptides show almost no differences in retention time under standard reverse-phase chromatography conditions. The derivatization of peptides with iodoacetic anhydride and 2-mercaptobenzimidazole proceeds rapidly and essentially to completion as illustrated in Figure 4 for Glu-fibrinopeptide. The ion at 1553 is the result of cyclization of the N-terminal Glu to form pyroglutamate in the Sigma preparation, which cannot react with the N-terminal reagents and consequently appears in all three spectra. Parent (Precursor) Ion Quantitation Scanning of a Complex Mixture. The PIQS approach was first tested using a sample of isotopically labeled BSA mixed with a tryptic digest of whole yeast protein extract to provide a complex background. The H4- and D4-labeled BSA samples were combined in a 60:40 ratio, and after clean up on a HILIC column, the fractions containing the main peptide elution peak were combined and concentrated. The total ion intensity for the yeast ions was, on average, roughly 30-fold higher for yeast (Figure 5), which is in line with the calculated molar ratio of about 20:1, yeast-to-BSA in the sample. Next, isotopically labeled 60:40 (H4/D4) BSA was mixed with 50:50-labeled ADH to make a mixture of labeled peptides, with the ADH peptide pairs of equal ion intensity, and only the BSA peptide pairs showing a difference in intensity. This was to demonstrate that no peptide pairs would be selected which fulfilled the isotopic separation criteria only and, furthermore,

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

that only pairs with a specific difference in intensity ratio would be chosen. In addition, a tryptic digest of whole yeast protein extract was added to provide complexity. Approximately, equal molar amounts of labeled BSA and ADH together with roughly 20-fold molar excess of yeast were mixed. The samples were cleaned using HILIC cartridges to remove excess and unreacted reagents after labeling. To select only the BSA-labeled peptides, a program was developed in ThermoFinnigan Instrument Control Language (ICL) for the Quantum Triple Stage Quadrupole mass spectrometer. The mass spectrometer is operated in parent (precursor) ion scanning mode, taking alternate scans of the parents of the signature ions at 191 and 195. The program monitors the parent ion masses and tracks the appearance of isotopically labeled ion pairs. Once a pair of parent ions has been registered for five consecutive scans with an intensity ratio fold change of (1.5 (for 60:40 H4/D4-labeled peptides), the mass spectrometer switches to MS/MS mode selecting first the H4 then the D4 ion and then reverts to parent ion scanning mode. The collision energy is set to increase in a linear fashion from a low to a high m/z value during MS/MS, ion pairs selected for fragmentation are stored in memory, and these masses are excluded 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 the absence of yeast peptides gave 13 positive peptides identified using the MASCOT search engine compared to only 10 peptides when using unlabeled BSA under the same chromatographic conditions (Table 3). This is probably the result of an increase in signal intensity when a basic charge is introduced by the label at the N-termini.

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

H4 parent (m/z)

D4 parent (m/z)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

315.4 428.5 632.1 554.7 641.2 462.5 368.4 420.4 654.6 512.4 517 449.5 662.7 664.7 646.8 559.6

317.3 430.5 634.2 556.6 643 464.5 370.4 422.4 656.7 514.5 519 453.5 664.7 668.7 648.8 561.7

H4/D4 ratio (%)

63/36 59/41 63/37 62/38 80/20 61/39 59/41 63/37 70/30 63/37 39/61 61/39 59/41 58/42 65/35 65/35

ion charge

sequence

2 2 2 2 2 2 2 2 2 2 2 1 2 1 2 2

? ? false pos. SLGKVGTR EKVLTSSAR VLTSSAR VASLR IETMR false pos. false pos. LCVLHEK *GGAG ? ? ALKAWSVAR YLYEIAR

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

The PIQS program selected 16 peptide pairs when analyzing the sample containing the mix of 60:40 H4/D4-labeled BSA in a 20-fold excess of whole yeast protein tryptic digest (Table 4). Despite the very complex yeast peptide background, 9 of the pairs were identified by both the heavy and the light MS/MS spectra using the MASCOT search engine. Of the 9 pairs, 8 were identified as BSA peptides and 1 pair was identified as belonging to an unknown B. taurus protein (MGC:127846). Thus, this Journal of Proteome Research • Vol. 6, No. 3, 2007 1111

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WoÄ hlander et al.

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

appears to be a reasonable number of positive identifications, especially given that not all the fractions from the HILIC separation were combined. The elution profile of the isotopically labeled peptides (Figure 6) shows that the difference between light- and heavy-labeled peptides is very small, possibly due to the large π electron cloud of the aromatic rings which interact predominantly with the C18 phase and masks the hydrogen and deuterium atoms at the edge of the ring. The isotopic ratios calculated by the program agreed well with the expected 60:40 (H4/D4) ratio for almost all peptides (Table 4), with only two exceptions. These incorrect ratios were caused by very early or late sampling in the elution profile of the ion pair spectra, where signal intensities are unstable. The remaining 8 peptide pairs failed to be matched, but upon manual inspection, only four of them could be defined as true “false positives”. These four showed either the presence of only very weak signature ions and/or pairs of fragmentation spectra that did not correlate with each other. The remaining four pairs showed spectra that correlated well and showed strong signature ions; however, the MASCOT searches gave no positive identifications. Because the PIQS label produces spectra dominated by b-ions rather than the more common y-ion series, this could be a reason for lower recognition and scoring in MASCOT. From the sample containing both 60:40 (H4/D4) labeled BSA and 50:50 (H4/D4) labeled ADH in the 20-fold excess of yeast digest, the PIQS program selected 11 pairs of BSA and no ADH pairs (Table 5). In this experiment, a different approach was taken. The sample mix was subjected to a fast separation on a RP-column with fraction collection. Each fraction was then subjected to MS analysis using nanospray emitters. This eliminates the problem of time-dependent sampling and 1112


Table 5. BSA Peptide Pairs (60:40 H4/D4) Selected by the PIQS 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)

1 2 3 4 5 6 7 8 9 10 11

629.64 554.39 368.27 641.16 420.32 449.28 462.49 516.64 559.38 646.62 662.25

633.50 556.34 370.27 643.14 422.24 453.28 464.45 518.75 561.24 648.63 664.30

H4/D4 ratio (%)

78/22 62/38 61/39 60/40 62/38 62/38 61/39 61/39 61/39 60/40 61/39

ion charge

sequence

1 2 2 2 2 1 2 2 2 2 2

YTR SLGKVGTR VASLR EKVLTSSAR IETMR *GGAG VLTSSAR LCVLHEK YLYEIAR ALKAWSVAR ?

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

resulted in very accurate ratios and no ADH pairs incorrectly selected. Of the 11 pairs selected, 7 were correctly identified with MASCOT. Upon manual evaluation, two more could be identified, while the third pair, also unidentified in the previous experiment, again displayed well-correlating spectra and strong signature ion signal, but no match could be found in the database.

Conclusions The PIQS method offers some advantages and complements the gel-based approach to proteomics because it is rather nondiscriminatory against type or composition of proteins, and

research articles


by varying the proteases used, even membrane proteins are accessible. Through variation of the prelabeling experiments, the method can be made widely applicable; exchanging the linker reagent, for example, can enable study of post-translational modifications. Compared to the ICAT reagent, the PIQS label benefits both from increasing signal intensity of b-ions and the almost negligible chromatographic separation between the heavy and light isotope. It is not isobaric like the elegant iTRAQ label and cannot be multiplexed to the same degree; as yet, it does allow dynamic peptide selection in real time. Currently, the reagent is added to the N-terminal in a twostep procedure. However, now that we have selected reagents with the desired properties, a single stage reagent can be synthesized. The synthesis is simple, and the reagents are very cheap in comparison to those commercially available now. The usefulness of PIQS, in comparison to other labeling strategies, lies in the selectivity for peptides with differential isotope ratios, which, applied to biological samples, will allow the selection and fragmentation analysis exclusively of peptides from proteins displaying differential expression or posttranslational modification. This greatly reduces data complexity and sets it apart from, for example, the iTRAQ approach.14 The label is not restricted to selected amino acids only, as opposed to a number of other isotopic labeling schemes as ICAT9 and hence should give a satisfactory representation of the proteins in the analyzed sample. When online analysis is used, a few aberrant ratios can be encountered, but these can be almost completely eliminated using prefractionation together with disposable nanospray emitters such as the Advion system. The experiments presented in this paper show how PIQS can be used for targeted studies and works as a proof of principle of the method. One application that we are currently investigating is the use of PIQS for absolute quantitation using ‘proteotypic’ or signature peptides selected for proteins of interest in a sample. We are also working on the application of the PIQS approach to whole-proteome studies of biological samples. The analysis of such complex samples requires more prefractionation prior to the final dynamic analysis, but PIQS is appearing very useful. Our main current limitation is the lack of very sensitive mass spectrometer with a high duty cycle such as the Q-Trap from Sciex (Toronto, Canada). Abbreviations: PIQS, parent-ion quantitation scanning.

Acknowledgment. This work was supported by grants from the Knut and Alice Wallenberg Foundation, by a Swegene Postdoctoral program grant to F.L., the Swedish Strategic

Research Council to CREATE Health (P.J.), and from Blanceflor Boncompagni-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 Serum Albumin showing the expected and observed peptides highlighted are available. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) O’Farrell, P. H. J. Biol. Chem. 1975, 250, 4007-4021. (2) Gorg, A.; Weiss, W.; Dunn, M. J. Proteomics 2004, 4, 3665-3685. (3) Gygi, S. P.; Corthals, G. L.; Zhang, Y.; Rochon, Y.; Aebersold, R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9390-9395. (4) Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19, 242-247. (5) Simpson, D. C.; Smith, R. D. Electrophoresis 2005, 26, 1291-1305. (6) Ishii, D.; Hibi, K.; Asai, K.; Nagaya, M.; Mochizuki, K.; Mochida, Y. J. Chromatogr. 1978, 156, 173-180. (7) Goshe, M. B.; Smith, R. D. Curr. Opin. Biotechnol. 2003, 14, 101109. (8) Julka, S.; Regnier, F. E. Briefings Funct. Genomics Proteomics 2005, 4, 158-177. (9) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (10) Zhou, H.; Ranish, J. A.; Watts, J. D.; Aebersold, R. Nat. Biotechnol. 2002, 20, 512-515. (11) Yao, X.; Freas, A.; Ramirez, J.; Demirev, P. A.; Fenselau, C. Anal. Chem. 2001, 73, 2836-2842. (12) Ong, S. E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M. Mol. Cell. Proteomics 2002, 1, 376-386. (13) Ong, S. E.; Kratchmarova, I.; Mann, M. J. Proteome Res. 2003, 2, 173-181. (14) Ross, P. L.; Huang, Y. N.; Marchese, J. N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.; Pappin, D. J. Mol. Cell. Proteomics 2004, 3, 1154-1169. (15) Gygi, S. P.; Rochon, Y.; Franza, B. R.; Aebersold, R. Mol. Cell. Biol. 1999, 19, 1720-1730. (16) Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol. 2001, 19, 379382. (17) Gevaert, K.; Van, Damme, J.; Goethals, M.; Thomas, G. R.; Hoorelbeke, B.; Demol, H.; Martens, L.; Puype, M.; Staes, A.; Vandekerckhove, J. Mol. Cell. Proteomics 2002, 1, 896-903. (18) Gevaert, K.; Goethals, M.; Martens, L.; Van, Damme, J.; Staes, A.; Thomas, G. R.; Vandekerckhove, J. Nat. Biotechnol. 2003, 21, 566569. (19) Carr, S. A.; Huddleston, M. J.; Bean, M. F. Protein Sci. 1993, 2, 183-196. (20) Munchbach, M.; Quadroni, M.; Miotto, G.; James, P. Anal. Chem. 2000, 72, 4047-4057.

PR0604312


Development of Reagents for Differential Protein Quantitation by

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