Reproducibility of Tryptic Digestion Investigated by Quantitative Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Charlotte Hagman,† Margareta Ramstro1 m,‡ Maria Jansson,§ Peter James,§ Per Håkansson,† and Jonas Bergquist*,‡ Division of Ion Physics, The Ångstro¨m Laboratory, Uppsala University, Box 534, SE-75121 Uppsala, Sweden, Department of Chemistry, Analytical Chemistry, Uppsala University, Box 599, SE-75124 Uppsala, Sweden, and Department of Electrical Measurements, Lund University, Box 118, SE-22100 Lund, Sweden Received October 21, 2004
In this study, the reproducibility of tryptic digestion of complex solutions was investigated using liquid chromatography Fourier transform ion cyclotron resonance (LC FT-ICR) mass spectrometry. Tryptic peptides, from human cerebrospinal fluid, (CSF) were labeled with Quantification-Using-EnhancedSignal-Tags (QUEST)-markers, or 1-([H4]nicotinoyloxy)- and 1-([D4]nicotinoyloxy)-succinimide ester markers. The analysis was performed on abundant proteins with respect-to-intensity ratios and sequence coverage and obtained by comparing differently labeled components from one or different pools. To interpret the dynamics in the proteome, one must be able to estimate the error introduced in each experimental steps. The intra sample variation due to derivatization was approximately 10%. The inter sample variation depending on derivatization and tryptic digestion was not more than approximately 30%. These experimental observations provide a range for the up- and down-regulations that are possible to study with electrospray ionization LC FT-ICR mass spectrometry. Keywords: cerebrospinal fluid • LC FT-ICR mass spectrometry • QUEST-markers • quantification • tryptic reproducibility • 1-([H4]nicotinoyloxy)- and 1-([D4]nicotinoyloxy)-succinimide ester markers
Introduction There are mainly two strategies to identify proteins and peptides in complex biological mixtures with mass spectrometry; either by fragmenting intact proteins with mass spectrometric methods1 or through proteolytic digestion followed by fragmentation. The later approach is referred to as “shot gun” proteomics and the identification takes place by matching detected peptide sequences with known sequences from databases, also called peptide mass fingerprinting.2-5 Sometimes the identification of a protein takes place by using accurate mass tag (AMT)s. An AMT is a peptide which acts as a characteristic tag for a protein or peptide since its mass can easily be distinguished from other peptides in the same mixture.6,7 The proteolytical digestion in proteome studies can take place with one2-7 or multiple proteases.8 Different proteases have different specificity, for example trypsin cuts at arginine and lysine residues. These two amino acids are among the most frequent ones in protein and peptides, and trypsin is therefore often used in proteomic studies. The enzyme activity is dependent on factors such as temperature, pH and the ratio between the substrate and enzyme.9 * To whom correspondence should be addressed. Fax: +46 18 471 3692. E-mail:
[email protected]. † Division of Ion Physics, The Ångstro¨m Laboratory, Uppsala University. ‡ Department of Chemistry, Analytical Chemistry, Uppsala University. § Department of Electrical Measurements, Lund University.
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Journal of Proteome Research 2005, 4, 394-399
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In several studies in our laboratory, we have taken advantage of the ultrahigh resolution and high sensitivity of the FT-ICR mass spectrometer. In well-calibrated spectra, the mass measurement errors are within a few ppm, and it is possible to identify peptides with high probability using only the m/z value. The approach involves simultaneous proteolytic digestion of all proteins in the sample, followed by a HPLC- or CEseparation step and finally identification by mass measurement.10-13 The systematic study of protein properties such as structure, function, interactions with other proteins, and comparison between systems in health and disease is defined as proteomics.14,15 The up- and down-regulations of certain proteins and peptides in CSF have been correlated to disease states. Thus, it is of great importance to develop methods to study these processes.16-18 Each protein has its own expression- and turnover-rate, so there is a dynamics in terms of expressed amount.19-21 In many proteomics studies, not only a single sample is analyzed, but complex tryptic protein mixtures from several tryptic digest events are investigated.22,23 It is thus important to be able to determine the error introduced by differences in the trypsin reproducibility and derivatization with global markers for quantitative analysis. In cerebrospinal fluid (CSF), as well as in other clinically interesting body fluids, half of the total protein content originates from human serum albumin, (HSA).24 Biologically more interesting proteins are often present in very small amounts and can therefore be suppressed in the ionization process. Thus, schemes to deplete 10.1021/pr049809r CCC: $30.25
2005 American Chemical Society
Reproducibility of Tryptic Digestion using FTICR-MS
research articles
Figure 1. Chemical structures of the global markers used in this study. The peptide mass shift after labeling is shown for each marker. In (a) SMTA, (b) SMTP, (c) [H4], and in (d) [D4].
high abundant proteins have been examined.25 One concern with this manipulation of the sample is the introduction of irreproducible losses. In this study, the reproducibility of tryptic digestion of cerebrospinal fluid, (CSF), was investigated by quantitative analysis of a few abundant proteins in the complex mixture. The quantitative analysis was performed with LC FT-ICR mass spectrometry.26 The tryptic protein mixtures of CSF were labeled with Quantification-Using-Enhanced-Signal-Tags (QUEST)-markers; the S-Methyl Thioacetimidate (SMTA)- and S-Methyl Thiopropionimidate (SMTP)-markers, or 1-([H4]nicotinoyloxy)-and 1-([D4]nicotinoyloxy)-succinimide ester markers. The SMTA- and SMTP-markers will react with the N-terminal amino group and the -amino group of the lysine residues. The [H4]- and [D4]- markers will react with the N-terminal amino group.27,28 Both markers react with a chemical group present on every peptide, therefore the labeling is referred to as global. Labeling an amino group with the SMTA- and SMTPmarker, results in a mass shift of 41.0302 and 55.0422 Da, as shown in Figure 1, parts a and b. The [H4]- and [D4]-labeled peptide will shift in mass with 106.0293 and 110.0341 Da, respectively (see Figure 1, parts c and d). The objective of this study was to use these two markers for quantitative analysis and to isolate the error due to tryptic digestion and derivatization. In quantitative analysis, the relative ion abundance of differently labeled pools detected in the mass spectrometer are correlated to concentration differences of the samples. The most commonly known label is ICAT, which has specificity for cysteine residues.29,30 The advantage of using global markers is that they will react with a chemical structure present in every part of the trypic digest, thus resulting in an increased probability for high sequence coverage. In ongoing projects, we are analyzing up- and downregulations of proteins and peptides of clinical relevance, using relative quantitative aspects. To be able to interpret an increased or decreased intensity ratio with LC FT-ICR MS, an estimation of the error introduced in each experimental step is required.
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 as 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. Synthesis of 1-([H4]nicotinoyloxy), ([H4]), and 1-([D4]nicotinoyloxy), ([D4]), Succinimide Esters. Further on these two markers are referred to as [H4] and [D4]. See ref 29 for further information.
Experimental Section
Sample Preparation. The CSF used in this study was taken from a pool consisting of >200 individual CSF samples drawn from patients in the age of 16-65 years. The samples were taken by lumbar puncture for diagnostic purposes, and the patients showed no signs of neurological or psychiatric disorders. Routine CSF analysis revealed no signs of inflammation or damage to the blood-brain barrier function.The study was approved by the Human Ethics Committee at the Faculty of Medicine, Sahlgrenska University Hospital, Go ¨ teborg, Sweden. Three samples of human cerebrospinal fluid (CSF) were prepared for analysis. Each sample, 300 µL, were centrifuged to dryness using a Speedvac system ISS110 (Savant Holbrook, N. Y., USA). The three remaining CSF-protein- and peptidepellets, with a total protein content of 180 µg, were separately dissolved in 100 µL 0.4 M NH4HCO3 and 8 M urea. The total protein concentration of each sample was determined using the bicinchoninic acid protein assay reagent (Pierce Chemical Company, Rockford, USA). Then 10 µL of 0.45 mM of dithiothreitol (Amersham Bioscience, Uppsala, Sweden) was added to each Eppendorf tube. The dissolved CSF-pellets were heated to 50 °C and incubated for 15 min. Then the mixture was cooled to ambient temperature and 10 µL 100 mM of iodoacetamide (Sigma-Aldrich) was added and this mixture was kept in darkness for 15 min. Trypsin (1418475) (Roche Diagnostics GmbH, Penzberg, Germany), 6.5 µg, was dissolved in 130 µL deionized water and added to each sample (resulting in 3.6% w/w (trypsin/protein)). The digestion was performed at 37 °C overnight in darkness.
Materials. Thioacetamide, 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 the procedure of Beardsley et al.,27 0.55 g thioacetamide was dissolved and stirred in 50 mL anhydrous dietyl ether 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.
Amidination of CSF Digests. SMTA was dissolved in 250 mM tris(hydroxymethyl)aminomethan to 130.2 g/L. Then 40 µL 0.73 mg/mL tryptic CSF was mixed with 40 µL dissolved SMTA and left at ambient temperature for 1 h. SMTP was dissolved in 250 mM tris(hydroxymethyl)aminomethan to 138.6 g/L. The same volume and concentration as above of tryptic CSF was mixed with 40 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; therefore dissolving the crystals in tris-buffer should be performed directly before Journal of Proteome Research • Vol. 4, No. 2, 2005 395
research articles labeling. The SMTA- and SMTP-labeled peptides were stored in -20 °C and no sign of degradation was observed over several months. [H4]- and [D4]-Labeling of CSF Digests. The [H4]- and [D4]markers were dissolved in N,N-Dimethylformamide, Sigma Aldrich (Steinheim, Germany), and ACN Sigma Aldrich (St. Louis, MO), respectively, to a final concentration of 5 mM. Digested CSF was mixed with an equal volume of 200 mM MOPS-buffer on ice to a final volume of 8 µL. Then 2 µL of dissolved marker, either [H4]- or [D4]-marker, was added to the tryptic CSF mixture. The reaction was allowed to continue for 1 h on ice. Desalting. Salts and other contaminants were removed by using a reversed-phase Zip-TipC18 pipet tip. The procedure was described previously by Hagman et al.26 Mass Spectrometry. A 9.4-T BioAPEX-94e FT-ICR mass spectrometer with a passively shielded magnet (Bruker Daltonics, Billercia, MA) was used in all MS experiments. The online electrospray was performed using a Black Dust coated electrospray emitter with an i.d. of 50 µm. The ions were accumulated in the hexapole for 600 ms prior to injection to the mass analyzer. Data were collected on a computer running Xmass in the chromatography mode. Datasets of 150-256 spectra were generated, where each spectrum of 256 K data points was collected during 10 s. All spectra were calibrated using abundant labeled CSF-peptide peaks. In experiments where the tryptic digest of CSF was labeled with SMTA-and SMTP-markers the calibration was performed with respect to the following known labeled HSA peptide masses: (m/z 574.6713, 584.0126, 712.8512, 726.8632, 870.4102, 884.4222, 1054.6292, 1068.6412) to adjust the calibration parameters. In the corresponding experiments with [H4]- and [D4]-markers, the following known labeled HSA peptide masses were used: (m/z 559.8141, 561.8267, 724.332, 726.3445, 1118.6210, 1122.6461, 1447.6557, 1451.6818). These m/z-values were chosen since they are evenly distributed with regard to retention time and numerical value. A quadratic fit to the second-order calibration equation m/z ) a/f + b/f2 + c, where f is the frequency and a, b, and c are free variables were used. After calibration the experimental m/z-values differed by less than 3 ppm from the theoretical values. High Performance Reversed-Phase Liquid Chromatography. The HPLC-separation was performed on an in-house packed C8-column, i.d. 200 µm, length 10 cm. The packing material was Nucleosil 300-10 C8 with a particle diameter of 10 µm manufactured by Macherey-Nagel (Du ¨ ren, Germany). Two HPLC-pumps (JASCO 1580; Tokyo; Japan) delivered a mobile phase gradient using solvents A and B. The solvent composition of A was as follows: ACN:H2O:HAc (5:94.5:0.5 v:v: v), and B consisted of the following: ACN:H2O:HAc (94.5:5:0.5 v:v:v), and the program of choice was: solvent A for 10 min, 100-50% A in 35 min and 50-0% A in 15 min. 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). After splitting, the flow rate over the column was approximately 1.8 µL/min. Data Analysis. Software for the analysis of complex spectra was written in C, MATLAB and Visual Basic script. The peaks in the mass spectra were sorted into isotopic clusters, the monoisotopic mass and the total intensity of each cluster were determined. The experimental peptide masses were compared to calculated masses of completely labeled tryptic peptides of the proteins of interest. If the masses agreed within 10 ppm, 396
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Figure 2. CSF was digested and labeled according to the schematic representation above. The [H4]- and [D4]-markers and the QUEST-markers, SMTA and SMTP, are global markers for quantitative analysis.
they were included in the lists for light or heavy markers, respectively. The two lists were then compared, and a labeled pair was found if the same peptide was present in both lists, the difference in elution time was
