Quantitative Analysis of Tryptic Protein Mixtures Using Electrospray

Feb 25, 2004 - Margareta Ramstr m , Igor Ivonin , Anders Johansson , H kan Askmark ... Roman Zubarev , Per H kansson , Sten-Magnus Aquilonius , Jonas ...
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Quantitative Analysis of Tryptic Protein Mixtures Using Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Charlotte Hagman,† Margareta Ramstro1 m,‡ Per Håkansson,† and Jonas Bergquist*,‡ Department of Engineering Sciences, Division of Ion Physics, Uppsala University, and Institute of Chemistry, Department of Analytical Chemistry, Uppsala University Received December 1, 2003

For the first time, quantitative analysis of tryptic protein mixtures, labeled with Quantification-UsingEnhanced-Signal-Tags (QUEST)-markers, were performed with electrospray ionization and a 9.4 T Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometer. Coupling a High-Pressure Liquid Chromatography (HPLC) separation step prior to mass analysis resulted in an increased amount of identified labeled tryptic peptides. The range for the determined intensity ratios of two peptides in a labeled pair was large, but the obtained median intensity ratio correlated very well with the corresponding concentration ratio. This method can be used for observing protein dynamics in a specific cell type, tissue, or in body fluids. Keywords: QUEST-markers • quantification • electrospray ionization • HPLC • FTICR mass spectrometry • complex samples

Introduction The protein expression is a dynamic process depending on the cell type, the cell cycle, and responses to genetic factors or pharmacological treatment. Previously, these changes have been studied by quantifying the amount of mRNA.1,2 That approach has its weakness since the correlation between the amount of mRNA and the functional protein is not always strong.3 The protein activity can be changed through posttranslational modifications, PTMs, such as phosphorylation and glycosylation. Phosphorylation is highly important for folding the protein into a functional form and regulates cellular events such as replication, transcription, translation, and signal transduction.4,5 Due to the variety of carbohydrate structures the glycosylation results in an increased diversity and specificity of the protein function.6,7 Most genes are thought to be regulated at multiple levels but the most common regulatory step is the transcriptional control.8 The protein expression is either up- or down-regulated as a result of this control step. Changes in the protein expression have been correlated with disorders both in terms of structure and regulating features. The human body fluids are sources of biological markers, in e.g., cerebrospinal fluid (CSF) biological markers for schizophrenia,9 Alzheimer’s disease,10-14 vascular dementia,11,12 and Creutzfeldt-Jakob disease,14-16 have been determined. * To whom correspondence should be addressed. Assoc. Prof. Jonas Bergquist. Department of Analytical Chemistry, Institute of Chemistry, Uppsala University, Box 599 S-751 24 Uppsala. Fax: +46 18 471 3692. E-mail: [email protected]. † Department of Engineering Sciences, Division of Ion Physics, Uppsala University. ‡ Department of Analytical Chemistry, Institute of Chemistry, Uppsala University. 10.1021/pr034119t CCC: $27.50

 2004 American Chemical Society

Several methods for quantification analysis in combination with mass spectrometry have been presented during the last years. The general approach involves a reaction between the marker and a specific amino acid or with a chemical group present in every enzymatic digest. The most well-known method is Isotope-Coded Affinity Tags (ICAT).17,18 The ICATreagent consists of three elements; the affinity tag, a linker which can incorporate stable isotopes and a reactive group that has specificity for thiol-groups. The sample complexity is reduced since only cysteine containing peptides will be isolated and further analyzed. After the affinity selection step the analysis is often performed using the LC-MS/MS approach. The method permits separation using biochemical and immunological approaches which suites the analysis of low abundant proteins. The drawbacks are that the method fails to cover parts of the biomolecules that lack cysteines, which has implications for the identification of proteins and peptides using database searches. The likelihood for a true match is reduced since the biological material for analysis is selected. Another method is Mass-Coded Abundance Tagging (MCAT). The lysine residue is converted into a homoarginine residue through guanidation.19-21 The proton affinity is increased after derivatization and thus the ionization efficiency of the analyte.23 The enhanced protonation results in a higher number of detected peaks, that is of high importance using databases for protein identification. The drawback is that ionization efficiencies for the untreated and treated samples are different and a comparison between the two pools will hence be difficult to interpret from a quantitative point of view. An additional approach is to incorporate stabile isotopes as internal standards to obtain quantitative information. The isotopes are either introduced by using different isotopes in the enzymatic digest Journal of Proteome Research 2004, 3, 587-594

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research articles step,24-26 or for bacterial proteome studies the isotopes are incorporated in vivo.26 Another approach is to convert carboxylic acids to their corresponding methyl ester using either d0 or d3- methanol.27 The main drawback of isotope-enriched reagents is the high cost. In this paper, we used a technique for comparative proteomics based on Quantification-Using-Enhanced-Signal-Tags (QUEST) markers developed by Beardsly et al.28 The two markers are S-Methyl Thioacetimidate (SMTA) and S-Methyl Thiopropionimidate (SMTP). Through amidination the markers are covalently linked to the N-termini and lysine residues in the tryptic protein digest. The advantage of using the QUESTmarkers is that they will react with a chemical structure present in every part of the trypic digest, thus resulting in global labeling. An increased proton affinity of the labeled tryptic peptides results in improved ionization efficiency.23 Since no selection of the biological material is applied prior to the analysis the requirements for the mass spectrometric method are specific. One mass spectrometric technique which satisfies the requirements is Fourier Transform Ion Cyclotron Resonance, (FTICR), mass spectrometry. FTICR provides a wide mass range, ultrahigh resolving power and excellent mass accuracy.29,30 The 9.4 T magnet provides a high magnetic field which leads to improvement of FTICR parameters such as: signal-to-noise level, dynamical range, mass selectivity, and resolution.31 The accurate mass determination affects the reliability of the protein identification since it allows more precise settings using databases for protein identification,32 and thus reducing the risk of miss matching. Electrospray ionization33 coupled to FTICR mass spectrometry has been used for characterization and identification of proteins and peptides in several studies.34-39 Through electrospray ionization, it is possible to generate multiply charged ions, and hence it is possible to analyze large biomolecules even though the FTICR mass analyzer has a limited m/z range.40,41 The electrospray response is linear over 2 to 3 orders of magnitude centered at 1 µM.42,43 It is the hydrophobicity of the analyte that causes the droplet surface affinity and results in increased chargeability.44-46 Therefore, labeling the analyte with markers, which contains hydrophobic components, should enhance the ionization. The stability of the whole experimental procedure was tested by running several consecutive runs and subsequently determining the sequence coverage. The average intensity ratio for different concentration ratios was determined as well as the standard deviation. A HPLC-separation with a C8-column was used to reduce suppression effects and the numbers of identified labeled pairs were compared to the results obtained from direct infusion experiments. The median for the intensity ratio of the labeled pair was used to quantify differences in protein concentration on a relative scale.

Experimental Section Materials. Tioacetamide, iodoacetamide, thiopropionamide, acetonitrile, iodomethane, trifluoroacetic acid, anhydrous dietyl ether, and acetone was purchased from Sigma Aldrich (St. Louis, MO). Synthesis of S-Methyl Thioacetimidate (SMTA). Following to the procedure of Beardsley et al.,28 0.55 g tioacetamide was dissolved and stirred in 50 mL anhydrous dietyleter at ambient temperature for 60 min. Then 440 µL iodomethane was added and the mixture was allowed to evaporate overnight in a fume hood. The obtained light yellow crystals were stored at ambient temperature in a vacuum chamber. 588

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Synthesis of S-Methyl Thiopropionimidate (SMTP). Thiopropionamide, 0.09 g, was dissolved in 5 mL acetone. The mixture was heated to 60 °C in a water bath and 190 µL iodomethane was added. After 1 h in the water bath the solution was allowed to evaporate overnight in the fume hood. Brown crystals were obtained after evaporation and were stored at ambient temperature in a vacuum chamber. Sample Preparation. Bovine serum albumin (BSA) (66397.9 Da), myoglobin (16951.5 Da from bovine heart), cytochrome C (from pig, 11565 Da) were obtained from Sigma-Aldrich. Trypsine, sequencing grade from bovine pancreas, came from Roche Diagnostics GmbH (Penzberg, Germany). BSA, 190 µg, was dissolved in 100 µL 0.4 M NH4CO3, 8 M urea and 10 µL of 0.45 mM of dithiothreitol (Amersham Bioscience, Uppsala, Sweden) was added. The mixture was heated to 50 °C and incubated for 15 min. Then the mixture was cooled to ambient temperature and 10 µL 100 mM of iodoacetamide (SigmaAldrich) was added and this mixture was kept in darkness for 15 min. Trypsin, 6.84 µg, was dissolved in 130 µL deionized water (resulting in 3.6% w/w (trypsin/protein)) and added to the mixture. The digestion was performed at 37 °C overnight in darkness. Amidination of Trypic Protein Digest. SMTA was dissolved in 250 mM tris(hydroxymethyl)aminomethan to 43.4 g/L. Then 100 µL of 10-µM tryptic BSA was mixed with 100 µL dissolved SMTA and left at ambient temperature for 1 h. SMTP was dissolved in 250 mM tris(hydroxymethyl)aminomethan to 46.2 g/L. The same volume and concentration as above of tryptic BSA was mixed with 100 µL dissolved SMTP and left at ambient temperature for 1 h. The exchange reaction was quenched by adding 1.0% trifluoroacetic acid. Note that the SMTA- and SMTP-compounds are very instable in solution,47 therefore dissolving the crystals in tris-buffer should be performed directly before mixing with the digest. However, the SMTA- and SMTP-labeled peptides were stored in -20 °C and no sign of degradation was observed over time (month). Desalting. Salts and other contaminants were removed by using a reversed-phase Zip-TipC18 pipet tip. First the tip was wetted in 10 µL 50% acetonitrile (ACN) and then equilibrated with 10 µL 1% acetic acid (HAc). A volyme of 10 µL SMTA and SMTP-labeled BSA of various concentrations was adsorbed onto the reverse-phase media by 30 repeated cycles of sample loading. To remove salt from the tip it was washed with 1% acetic acid, total volume of 30 µL. Eluting the proteins for the tip was done with 10 µL 50% ACN in 1% HAc followed by 10 µL of 100% ACN. This procedure was repeated twice for every 10 µL of sample resulting in a total volume of 60 µL. After desalting, the eluate was vacuum centrifuged to dryness. For the direct infusion experiments the pellet was dissolved in 50:49:1%, H2O: Methanol: Acetic Acid. For the LC-experiments the pellet was dissolved in ACN: H2O: HAc (5:94.5:0.5 v:v:v). Mass Spectrometry. A 9.4-T BioAPEX-94e Fourier Transform Ion Cyclotron Resonance Mass Spectrometer with a passively shielded magnet (Bruker Daltonics, Billercia, MA) was used in all MS experiments. The direct infusion was performed using a Black Dust coated capillary with an i.d. of 50 µm.48 The ions were accumulated in the hexapole for 1000-1500 ms prior to injection to the mass analyzer. Ions were accumulated in the mass analyzer through the SIDE-KICK system. All spectra were calibrated using five to six abundant unlabeled BSA peaks (m/z 582.3189, 740.4013, 862.9209, 940.9642, 1163.6307, 1479.5354) to determine the calibration parameters. A quadratic fit to the second-order calibration equation was used.

Quantitative Analysis of Tryptic Protein Mixtures

High-Pressure Reversed Phase Liquid Chromatography. The separation was performed on an in-house packed C8column, i.d. 200 µm, length 10 cm. The packing material was Nucleosil 300-10 C8 with a particle diameter of 10 µm and it was manufactured by Macherey-Nagel (Du ¨ ren, Germany). Two HPLC-pumps (JASCO 1580; Tokyo; Japan) delivered a mobile phase gradient using solvents A and B. Prior to analysis the sample was dissolved in a volume of 20 µL of solvent A and was injected to the column using a six-port injector valve (Valco Instrument, Schenkon, Switzerland). The solvent composition of A was: ACN: H2O: HAc (5: 94.5: 0.05 v: v: v), and B consisted of: ACN: H2O: HAc (94.5: 5: 0.05 v: v: v). After splitting the flow rate over the column was approximately 1.8 µL /min. The separated peptides were monitored using a UVdetector prior to mass spectrometric analysis. The unlabeled BSA peaks (m/z 582.3189, 740.4013, 862.9209, 940.9642, 1163.6307, 1479.5354) were used to determine calibration parameters for internally calibrating the entire HPLC-FTICR MS mass spectra. These peaks were evenly distributed through the entire experiment both with respect to the retention times and m/z-values. Data Analysis. Programs were written in MATLAB to analyze the complex spectra. The programs included algorithms to reduce the peaklist to a list of isotopic clusters and to calculate the total intensity of the clusters as well as algorithms for matching the labeled enzymatic digest with the unlabeled enzymatic digest obtained from MS-Digest. A flow diagram is included in Figure 1. Briefly, the peaks in the mass spectrum were sorted into isotopic clusters, the monoisotopic mass and the intensity of each cluster was determined. The experimental peptide masses were compared to calculated masses of tryptic peptides of the proteins of interest labeled with SMTA- or SMTP-markers. If the masses agreed within a certain range (typically 5 to 20 ppm) they were included in the list for light or heavy markers, List 1 and List 2, respectively. To quantify the concentration differences, the two lists were compared and if the same number of markers and charges of a tryptic peptide were found in both lists, the total intensities of these matching peaks were divided and the quota was entered in List 3. Thus in List 3, each intensity ratio between the two peptides of a labeled pair was stored; the mean or the median of these intensity ratios was compared to the concentration ratio of the labeled pools.

Results and Discussion Initial experiments investigated electrospray ionization efficiency upon labeling of a tryptic protein digest with QUESTmarkers. The signal intensities of the unlabeled variant and the labeled variant of a specific tryptic fragment of cytochrome C originating from residues 56-73 were determined. The 56-73 residues fragment, (GITWGEETLMEYLENPKK), contained three possible labeling sites: two at the lysine residues and one at the N-terminal position. For the SMTA-labeled cytochrome C tryptic fragment 56-73, the total intensity was enhanced with a factor of 2.4, the corresponding intensity enhancement for the SMTP-labeled cytochrome C tryptic fragment was 1.5, see Figure 2A and 2B. Since the 56-73 residues fragment is triply charged, labeling with three markers will result in a QUESTlabeled pair of peaks that will be separated due to the presence of an extra CH2-group on the SMTP-markers. It has been reported for MALDI-experiments, that the average ionization efficiency of some selected QUEST-labeled cytochrome C tryptic peptides was enhanced with a factor of 1.5-7.4.28 The

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Figure 1. Overview of the computer algorithm used for the identification of labeled tryptic peptides. The intensity ratio between the two labeled peptides in a labeled pair was determined.

average signal gain was correlated to the numbers of possible labeling sites and the size of the tryptic fragment. A tryptic peptide from cytochrome C containing 15 amino acids and two lysine residues labeled with SMTA increased the signal with a factor of 1.5.28 The result obtained in our study with electrospray ionization, an enhanced signal by a factor of 2.4, was consistent with the signal enhancement obtained with MALDI. The signal enhancement in the electrospray experiments could be correlated to both the increased hydrophobicity,44-46 and increased gas-phase basicity.28 The analysis, including identification of labeled tryptic protein fragments and calculation of the intensity ratio between the two peptides in a labeled pair, was done with a MATLABalgorithm, see Figure 1.The reproducibility of the results using ESI-FTICR MS to analyze labeled tryptic digests was investigated. The concentration ratio for SMTA- and SMTP-labeled BSA was 1:2 and the total sample concentration was kept constant at 5.5 µM. The tryptic digest of the 66.4 kDa BSA corresponds in complexity to a mixture of smaller digested proteins. Four consecutive experiments with the same settings were recorded and each experiment was performed during 64 Journal of Proteome Research • Vol. 3, No. 3, 2004 589

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Figure 2. A. Two first masses in the isotopic distribution are originating from the triply charge 57-76 residues peptide fragment of unlabeled cytochrome C. The sample concentration was 2 µM and the spectrum was collected during 64 s. B. SMTA- and SMTPlabeled cytochrome C were mixed to a concentration of 4 µM. The spectrum was collected during 64 s. The signal enhancement was calculated using the total intensity of the isotopic distribution from the labeled and unlabeled cytochrome C. The two experiments were performed with the same flow rate and electrospray settings.

Figure 3. Mean value and standard deviation of the sequence coverage of the SMTA-labeled BSA, the SMTP-labeled BSA and the sequence coverage for peptides labeled with both labels was determined. Four consecutive runs with the same settings were recorded. Each experiment was performed during 64 s.

s. The sequence coverage for the SMTA- or SMTP-labeled BSA, as well as the sequence coverage of BSA labeled with both markers, were determined. When tolerating a mass measurement error of 10 ppm, the sequence coverage for SMTA-labeled BSA was determined to 0.46 ( 0.002, (mean ( SD), n ) 4) and the corresponding SMTP-labeled BSA the sequence coverage was 0.55 ( 0.024. The sequence coverage of BSA labeled with both markers was determined to 0.29 ( 0.020, see Figure 3. The small standard deviation suggests that the fluctuations of the electrospray are minor and that the experimental setup is stable. In this experiment 22 SMTA-labeled tryptic BSA peptides and 49 identified SMTP-labeled tryptic BSA peptides were identified, see Figure 4A. We identified 13 labeled pairs of BSA, 4 of these hits were assigned for two charge states, see Figure 4B. For the 13 labeled pairs an analysis of the total intensity ratio was performed. The obtained ratio is used to relatively 590

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quantify the concentration difference between the SMTA- and SMTP-labeled BSA. The average intensity ratio was determined to 0.76 ( 0.60, the expected value was 0.5 since the SMTPlabeled BSA had twice as high concentration as the SMTAlabeled BSA. During mass analysis, using the FTICR mass spectrometer, the ions are trapped inside the mass analyzer. If the ions experience changes in the coulomb repulsion they will be pushed into larger orbits which results in small shifts in the detection-frequency and consequently the m/z-value. Therefore the calibration parameters in the different experiments but with the same total sample concentration can be considered as identical. To investigate the correlation between the sample concentration and obtained sequence coverage, the total concentration was reduced with a factor of 2 to 2.25 µM. Low sequence coverage would indicated that the concentration limit was reached. Several different concentration ratios SMTA/ SMTP with the same total concentration were investigated ranging from 1.38/1.38 to 2.29/0.46 µM, see Figure 5. The identified corresponding sequence coverage for the SMTAlabeled BSA at 20 ppm mass accuracy was 0.62-0.67. For the SMTP-labeled BSA the sequence coverage was determined to 0.65-0.34. The analysis of the SMTA-labeled BSA displayed the fact that an increase in concentration with a factor of 1.7 resulted in increased sequence coverage of 0.05. For the SMTP-labeled BSA the decrease in concentration with a factor of 3 resulted in a decrease of the sequence coverage by 0.31. The amount of labeled pairs of the digested BSA was consequently decreasing with the concentration of SMTP-labeled BSA. SMTA/SMTP-concentration ratios of 1 and 2 provide relatively high sequence coverage but for the concentration ratio of 5 the sequence coverage was less then 10%. The experimentally obtained intensity ratio of the assigned hits were plotted against the concentration ratio, see Figure 6. The intensity ratio is used as a measurement of the relative

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Quantitative Analysis of Tryptic Protein Mixtures

Figure 4. A. Sequence coverage of SMTA-labeled BSA was determined by 22 separate tryptic peptide matches (upper curve) along the sequence, for the SMTP-labeled BSA 49 separate matches were obtained (lower curve). B. The total number of hits from BSA labeled with both labels was 17 and the sequence coverage was determined to 0.30. Some parts of the sequence were identified by two hits that correspond to different charge states of the tryptic fragment of labeled BSA. For the assigned hits the intensity ratio between the SMTA- and SMTP-labeled BSA was determined to 0.76 ( 0.60 (mean ( SD, n ) 4)

Figure 5. Sequence coverage of SMTA- or SMTP-labeled BSA and labeled BSA with both markers is plotted as a function of the concentration ratio. In all three separate experiments the total BSA concentration was constant. [ ) SMTA-labeled BSA, 9 ) SMTP-labeled BSA, 2 ) SMTA and SMTP-labeled BSA.

quantity of differently labeled protein pools. The standard deviation for all intensity ratios is rather large in these experiments; this was also the case using the QUEST- labeling method in conjugation with MALDI.28 A further analysis of the direct infusion experiments showed that some of the identified labeled pairs were present in all five spectra originating from labeled tryptic fragments of BSA of masses 1304.709, 1442.635, 1906.913, respectively (see Table 1). The peaks which would correspond to labeled tryptic fragment 286-297 of BSA, mass 1906.913 Da, did not show any correlation to the increasing concentration ratio. The second labeled pair which was found in all spectra was originating from the 157-167 residues fragment of BSA, mass of 1442.635 Da. For the 157-167 residues fragment the intensity ratio obtained form the SMTA- and SMTP-labeled peptides for the concentration ratio 1, 2, and 5 were 0.66, 2.64, and 10.23, respectively. For the labeled peptides originating from the tryptic BSA peptide 402-412, mass of 1304.709 Da, the intensity ratio of the two labeled peptides were, 0.53, 1.82, 5.71, and in good agreement with the SMTA/SMTP-concentration ratio of 1, 2, 5, respectively. In the case where the intensity ratio for the assigned labeled pair was constant through all five experiments, the deviation could be explained by the presents of a compo-

Figure 6. Intensity ratio for SMTA/SMTP-labeled BSA, [, is plotted as a function of SMTA/SMTP concentration ratio, the theoretical value, 2, is also included. For the different concentration ratios the mean intensity ratio and standard deviation were determined and n equals the number of identified labeled pairs. For the concentration ratio of 1, the intensity ratio was 0.97 ( 0.71, n ) 21, the concentration ratio of 2 resulted in an intensity ratio of 3.38 ( 2.84, n ) 15. The concentration ratio of labeled SMTA/SMTP of 5 gave a calculated intensity ratio of 4.56 ( 3.67, n ) 6. Table 1. Three Labeled Peptide Pairs Were Found for All Concentration Ratiosa SMTA/SMTP unlabeled tryptic fragment from BSA

1304.709 1442.635 1906.913

concentration ratios 1

2

5

obtained intensity ratios 0.53 1.82 5.71 0.66 2.64 10.23 0.88 0.92 0.20

a The first pair provided a good correlation between the SMTA/SMTPconcentration ratio and the obtained mean value for the intensity ratio. The second pair had an intensity ratio which is growing exponentially along the concentration ratio. The intensity ratio of the third labeled pair is rather constant regardless of the concentration ratio.

nent that is not correlated to concentration differences such as contaminants. The presence of the other two types of deviating behavior, described above, gives altogether a good Journal of Proteome Research • Vol. 3, No. 3, 2004 591

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Figure 7. Magnification of the selected area from the 2D-plot shows the SMTA- and SMTP-labeling of the 1304.709 BSA tryptic fragment. The SMTA/SMTP ratio is 1 and a single label has been incorporated into the BSA fragment. The SMTA-labeled fragment is eluting in spectra 87 and the corresponding SMTP-labeled fragment appears in spectra 91.

explanation to the large standard deviation obtained in our experiments. Even though the FTICR provides high mass accuracy, false matches will become more frequent with increasing complexity of the spectra. Therefore, it is highly important that the identification of the up- or down regulation of proteins in complex samples is based on as many tryptic peptide fragments as possible. The peptides in this study are only identified by mass. To be able to analyze even more complex samples and lower abundant proteins an HPLC-system was coupled to the FTICR mass spectrometer. In the direct infusion experiments, approaching the concentration limit, the SMTA/SMTP-ratio provided low sequence coverage, see Figure 5. The total sample concentration was the same for the HPLC-experiments as used in the direct infusion experiments. The QUEST-labels have a hydrophobic component, which will result in an increase in ionization efficiency but also causes stronger interactions with the reversed phase packing material compared to the unlabeled tryptic peptide. The SMTP-label contains an additional CH2group compared to the SMTA-label and therefore labeled tryptic fragments have different retention times and will thus experience different electrospray conditions, see Figure 7. For the reversed phase HPLC-separation a C8-packing material was chosen since it is less hydrophobic then a C18-material. The total hydrophobicity of a peptide in a tryptic digest is a function of the amino acids composition and the numbers of labels. For the SMTA/SMTP-concentration ratio of 1, totally 113 hits were identified in the HPLC-FTICR-experiments. The elution time of the SMTA- and SMTP-labeled BSA depends on the amino acid composition of the tryptic fragment and the numbers of markers. The difference in retention time between the SMTA- and SMTP-labeled BSA fragments is plotted against the frequency of each difference, see Figure 8. The labeling with 592

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Figure 8. Retention time is dependent on the amino acid composition in the tryptic fragment as well as the numbers of labels. The difference in elution spectra of the SMTP-labeled BSA peptide to the corresponding SMTA-labeled peptide is plotted as a function of the frequency of each difference.

the QUEST-labels is not complete. Peptides that are labeled with the maximal number of markers will have longer retention time compared to the corresponding less labeled. The median for differences in retention time is 7 scans, which corresponds to approximately 70 s delay of the second peak. The SMTPlabeled peptides were always eluting after the corresponding SMTA-labeled peptide since they were more hydrophobic. In the direct infusion experiment, testing the reproducibility of the system, collected during 64 s, the standard deviation for the obtained sequence coverage in four separate experiments was minor, see Figure 2. Due to the effects of separation, different amount of sample was eluting at different time points affecting the performance of the electrospray. The HPLCseparation also results in a varying solvent composition during the experiment. Thus, the HPLC-FTICR experiments have a larger variance in the electrospray conditions compared to the direct infusion experiments.

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Quantitative Analysis of Tryptic Protein Mixtures Table 2. For Different Concentration Ratios the Numbers of Assigned Hits for SMTA- or SMTP-labeled BSA as Well as BSA Labeled with Both Markers Is Included in the Tablea SMTA/SMTP

SMTA/SMTP nos. of hits assigned intensity ratio concentration ratio SMTA SMTP matches median range

10 ppm 1 2 5

190 197 207

190 188 129

113 111 64

1.14 2.35 8.59

(0.04-16.27) (0.16-28.22) (1.75-173.7)

1 2 5

134 125 130

166 140 89

84 70 40

0.95 2.14 7.18

(0.05-16.27) (0.16-21.1) (1.75-173.7)

5 ppm

a The determined intensity ratio for the assigned hits is included and the range of the experimental data.

SMTA/SMTP-concentration ratios of 1, 2, and 5 were investigated at two levels of mass accuracy with respect to several parameters, see Table 2. The number of assigned hits for both SMTA- and SMTP-labeled BSA as well as the number of assigned pairs increased dramatically compared to the direct infusion experiments due to the reduction of ion suppression effects. The data range is large and therefore the mean value is not used. However, the median values for these different ratios are close to the theoretical values. The variation that could be seen in both the direct infusion experiments and in the HPLC-experiment partly depends on false matches. That phenomenon is increasing with the complexity of the sample but since the protein digest was not completely labeled, the number of false matches could probably be reduced if the unlabeled peaks were removed from the dataset. Regardless of large variations in retention times between the two peptides of a labeled pair, the median value for the intensity ratios shows that it is possible to use this method to study the protein dynamics in complex samples.

Concluding Remarks Labeling tryptic protein mixtures with QUEST-markers resulted in enhanced ionization efficiency. The intensity ratio, obtained from a labeled pair, is interpreted as a relative quantification of the protein concentration in differently labeled pools. For direct infusion experiments the mean values for the intensity ratios were close to the theoretical values but the standard deviations were large. Coupling a separation step prior to mass analysis reduced the ion suppression effects and resulted in an increased number of assigned labeled pairs. To reduce the effect of false matches, the median values were used to determine the intensity ratios obtained after coupling a separation step to the FTICR mass spectrometer. The difference in retention time between two labeled peptides in a labeled pair may imply varying electrospray conditions. However, the determined median values for the intensity ratios were in good agreement with the concentration ratio. The combination of HPLC-FTICR mass spectrometry and labeling tryptic protein mixtures with QUEST-markers showed promising results for quantifying proteins in even more complex biological samples.

Acknowledgment. The author wish to acknowledge: Johan Kjellberg for technical assistance, Ardeshir Amirkhani for preparing the LC column, Igor Ivonin for preparing the 2Dplot, Richard Beardsly for communication concerning the QUEST-markers. Financial support from Knut och Alice Wal-

lenberg, Swedish Research Council Grants 621-2002-5261, 6292002-6821 (J. B.), and K-1618-1999(P. H.). J. B. has a senior research position at the Swedish Research Council (VR).

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