Proteome analysis of a human heptocellular carcinoma cell line, HCC-M: an update.

Recently, we reported the proteome analysis of a human hepatocellular carcinoma cell line, HCC-M (Electrophoresis 2000, 21, 1787-1813), using two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). From a total of 408 unique spots excised from the 2-DE gel, 301 spots yielded good MALDI spectra. Out of these, 272 spots had matches returned from the database search leading to the identification of these proteins. Here, we report the results on the identification of the remaining 29 spots using nanoelectrospray ionization-tandem mass spectrometry (nESI-MS/MS). First, "peptide tag sequencing" was performed to obtain partial amino acid sequences of the peptides to search the SWISS-PROTand NCBI nonredundant protein databases. Spots that were still not able to find any matches from the databases were subjected to de novo peptide sequencing. The tryptic peptide sequences were used to search for homologues in the protein and nucleotide databases with the NCBI Basic Local Alignment Search Tool (BLAST), which was essential for the characterization of novel or post-translationally modified proteins. Using this approach, all the 29 spots were unambiguously identified. Among them, phosphotyrosyl phosphatase activator (PTPA), RNA-binding protein regulatory subunit, replication protein A 32 kDa subunit (RP-A) and N-acetylneuraminic acid phosphate synthase were reported to be cancer-related proteins.     

Proteomics based on selecting and quantifying cysteine containing peptides by covalent chromatography

This paper describes a procedure in which cysteine containing peptides from tryptic digests of complex protein mixtures were selected by covalent chromatography based on thiol-disulfide exchange. identified by mass spectrometry, and quantified by differential isotope labeling. Following disruption of disulfide bridges with 2,2'-dipyridyl disulfide, all proteins were digested with trypsin and acylated with succinic anhydride. Cysteine containing peptides were then selected from the acylated digest by disulfide interchange with sulfhydryl groups on a thiopropyl Sepharose gel. Captured cysteine containing peptides were released from the gel with 25 mM dithiothreitol (pH 7.5) containing 1 mM (ethylenedinitrilo)tetraacetic acid disodium salt and alkylated with iodoacetic acid subsequent to fractionation by reversed-phase liquid chromatography (RPLC). Fractions collected from the RPLC column were analyzed by matrix-assisted laser desorption ionization mass spectrometry. Based on isotope ratios of peptides from experimental and control samples labeled with succinic and deuterated succinic anhydride, respectively, it was possible to determine the relative concentration of each peptide species between the two samples. Peptides obtained from proteins that were up-regulated in the experimental sample were easily identified by an increase of the relative amount of the deuterated peptide. The results of these studies indicate that by selecting cysteine containing peptides, the complexity of protein digest could be reduced and database searches greatly simplified. When coupled with the isotope labeling strategy for quantification it was possible to determine proteins that were up-regulated in plasmid bearing Escherichia coli when expression of plasmid proteins was induced. Up-regulation of several proteins of E. coli origin was also noted.     

Mass spectrometric approaches for the characterization of proteins on a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer

This study demonstrates structural and conformational characterization of proteins by nanoflow electrospray ionization (nanoESI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) utilizing a quadrupole time-of-flight (Q-TOF) mass spectrometer (Micromass, Manchester, England). Model peptides were successfully sequenced at the 35 attomole (amol) level, and peptides derived from a tryptic in-gel digest of 25 femtomole (fmol) bovine serum albumin (BSA) were successfully sequenced. The results demonstrated that the MS/MS sensitivity of the Q-TOF clearly surpassed the detection limit of the silver stain. A silver destaining step greatly improved the mass analysis of peptides derived from in-gel digests. Interestingly, sequence analysis revealed BSA residue 424 (tyrosine) as a potential chlorination site. In addition, a modified procedure was successfully used to extract and measure the masses of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE)-resolved proteins in the 10-68.5 kDa range. The Q-TOF was also used to monitor conformational changes of proteins. These experiments demonstrated an acid-induced denaturation of BSA in the pH 3-4 range, and heat-induced unfolding of cytochrome c between 50 and 60 degrees C. Finally, Zn2+ binding was demonstrated for the carbonic anhydrase apoprotein. In summary, the wide range of applications and the high quality of the experimental data made the Q-TOF mass spectrometer a powerful analytical tool for protein characterization.     

Optimization of microfabricated nanoliter-scale solid-phase extraction device for detection of gel-separated proteins in low abundance by matrix-assisted laser desorption/ionization mass spectrometry

A nano-scale solid-phase extraction (SPE) device was developed for the detection of gel-separated proteins in low abundance by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with a simplified microfabrication technology. By using SU-8 photoresist instead of epoxy glue to connect the microchannel and transfer capillary, polymeric contaminant signals in MS analysis were significantly reduced. Micro SPE columns with different capacities and geometric characteristics were investigated in order to increase the detection sensitivity and decrease spot size for MALDI-TOF-MS analysis. It is shown that enhancements in sensitivities for the detection of proteins in low abundance were correlated with the reduction in column capacity and increase in column aspect ratio. Fifty nanoliters of matrix solution were sufficient to elute the sample completely from the optimized micro SPE column with 3.5 nL capacity. The mass spectrum of a 5 fmol in-gel tryptic digest of bovine serum albumin (BSA), processed by the micro SPE column, demonstrated that 29 peptides matched the protein giving a sequence coverage of 51%, which was better than that obtained from analysis of 25 fmol of the same sample prepared by the dried-droplet method. With the micro SPE column treatment of 2 microL of digestion supernatant of a gel spot of the IQGAP1 protein, 15 peptides were detected from the mass spectrum with the highest individual score of 111, while, with a ZipTip procedure, only nine peaks were detected with the highest individual score of 71. Analytical results demonstrated that this approach greatly improved the sequence coverage and identification specificity for the tested protein. It can serve as a very useful tool in proteomics studies, especially for low abundance proteins.     

Mass spectrometric characterization of mitochondrial electron transport complexes: subunits of the rat heart ubiquinol-cytochrome c reductase

Complex III of the mitochondrial electron transport chain, ubiquinol-cytochrome c reductase, was isolated by blue native polyacrylamide gel electrophoresis. Ten of the 11 polypeptides present in this complex were detected directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following electroelution of the active complex. Tryptic and chymotryptic digestion of the complex permit the identification of specific peptides from all of the protein subunits with 70% coverage of the 250 kDa complex. The mass of all 11 proteins was confirmed by second dimension Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and elution of the separated polypeptides. Additionally, the identity of the core I, core II, cytochrome c and the Rieske iron-sulfur protein were confirmed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) characterization of the peptides generated by in-gel trypsin digestion of the SDS-PAGE separated proteins. The methodology demonstrated for analyzing this membrane-bound electron transport complex should be applicable to other membrane complexes, particularly the other mitochondrial electron transport complexes. The MS analysis of the peptides obtained by in-gel digestion of the intact complex permits the simultaneous characterization of the native proteins and modifications that contribute to mitochondrial deficits that have been implicated as contributing to pathological conditions.     

Technical innovations for the automated identification of gel-separated proteins by MALDI-TOF mass spectrometry

The combination of gel-based two-dimensional protein separations with protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is the workhorse for the large-scale analyses of proteomes. Such high-throughput proteomic approaches require automation of all post-separation steps and the in-gel digest of proteins especially is often the bottleneck in the protein identification workflow. With the objective of reaching the same high performance of manual low-throughput in-gel digest procedures, we have developed a novel stack-type digestion device and implemented it into a commercially available robotic liquid handling system. This modified system is capable of performing in-gel digest, extraction of proteolytic peptides, and subsequent sample preparation for MALDI-MS without any manual intervention, but with a performance at least identical to manual procedures as indicated on the basis of the sequence coverage obtained by peptide mass fingerprinting. For further refinement of the automated protein identification workflow, we have also developed a motor-operated matrix application device to reproducibly obtain homogenous matrix preparation of high quality. This matrix preparation was found to be suitable for the automated acquisition of both peptide mass fingerprint and fragment ion spectra from the same sample spot, a prerequisite for high confidence protein identifications on the basis of peptide mass and sequence information. Due to the implementation of the stack-type digestion device and the motor-operated matrix application device, the entire platform works in a reliable, cost-effective, and sensitive manner, yielding high confidence protein identifications even for samples in the concentration range of as low as 100 fmol protein per gel plug.      

Automated neutral loss and data dependent energy resolved "pseudo MS3" for the targeted identification, characterization and quantitative analysis of methionine- containing peptides

A strategy involving the fixed-charge sulfonium ion derivatization, stable isotope labeling, capillary high- performance liquid chromatography and automated data dependent neutral loss scan mode tandem mass spectrometry (MS/MS) and "pseudo multiple mass spectrometry (MS(3))" product ion scans in a triple quadrupole mass spectrometer has been developed for the "targeted" gas-phase identification, characterization and quantitative analysis of low abundance methionine-containing peptides present within complex protein digests. Selective gas-phase "enrichment" and identification is performed via neutral loss scan mode MS/MS, by low energy collision-induced dissociation of the derivatized methionine side chain, resulting in the formation of a single characteristic product ion. Structural characterization of identified peptides is then achieved by automatically subjecting the characteristic neutral loss product ion to further dissociation by data dependent product ion scan mode pseudo MS(3) under higher collision energy conditions. Quantitative analysis is achieved by measurement of the abundances of characteristic product ions formed by sequential neutral loss scan mode MS/MS experiments from "light" ((12)C) and "heavy" ((13)C) stable isotope encoded fixed-charge derivatized peptides. In contrast to MS-based quantitative analysis strategies, the neutral loss scan mode MS/MS method employed here was able to achieve accurate quantification for individual peptides at levels as low as 100 fmol and at abundance ratios ranging from 0.1 to 10, present within a complex protein digest.     

Analysis of proteins copurifying with the cd4/lck complex using one-dimensional polyacrylamide gel electrophoresis and mass spectrometry: comparison with affinity-tag based protein detection and evaluation of different solubilization methods

Mass spectrometry-based identification of the components of affinity purified protein complexes after polyacrylamide gel electrophoresis (PAGE) and in-gel digest has become very popular for the detection of novel protein interactions. As an alternative, the entire protein complex can be subjected to proteolytic cleavage followed by chromatographic separation of the peptides. Based on our earlier report of a method using affinity tag-mediated purification of cysteine-containing peptides to analyse proteins present in an affinity purification of the CD4/lck receptor complex, we here evaluated the use of one-dimensional polyacrylamide gel electrophoresis for analysis of the same receptor complex purification. Using electrospray and tandem mass spectrometry analyses of tryptic peptides from in-gel digested proteins we identified the components of the CD4 receptor complex along with 23 other proteins that were all likely to be non-specifically binding proteins and mainly different from the proteins detected in our previous study. We compare the alternative strategy with the affinity tag-based method that we described earlier and show that the PAGE-based method enables more proteins to be identified. We also evaluated the use of a more stringent lysis buffer for the CD4 purification to minimise non-specific binding and identified 52 proteins along with CD4 in three independent experiments suggesting that the choice of lysis buffer had no significant effect on the extent of non-specific binding. Non-specific binding was inconsistent and involved various types of proteins underlining the importance of reproducibility and control experiments in proteomic studies.     

Optimized sample-processing time and peptide recovery for the mass spectrometric analysis of protein digests

Proteomics requires an optimized level of sample-processing, including a minimal sample-processing time and an optimal peptide recovery from protein digests, in order to maximize the percentage sequence coverage and to improve the accuracy of protein identification. The conventional methods of protein characterization from one-dimensional or two-dimensional gels include the destaining of an excised gel piece, followed by an overnight in-gel enzyme digestion. The aims of this study were to determine whether: (1) stained gels can be used without any destaining for trypsin digestion and mass spectrometry (MS); (2) tryptic peptides can be recovered from a matrix-assisted laser desorption/ionization (MALDI) target plate for a subsequent analysis with liquid chromatography (LC) coupled to an electrospray ionization (ESI) quadrupole ion trap MS; and (3) an overnight in-gel digestion is necessary for protein characterization with MS. These three strategies would significantly improve sample throughput. Cerebrospinal fluid (CSF) was the model biological fluid used to develop these methods. CSF was desalted by gel filtration, and CSF proteins were separated by two-dimensional gel electrophoresis (2DGE). Proteins were visualized with either silver, Coomassie, or Stains-All (counterstained with silver). None of the gels was destained. Protein spots were in-gel trypsin digested, the tryptic peptides were purified with ZipTip, and the peptides were analyzed with MALDI and ESI MS. Some of the samples that were spotted onto a wax-coated MALDI target plate were recovered and analyzed with ESI MS. All three types of stained gels were compatible with MALDI and ESI MS without any destaining. In-gel trypsin digestion can be performed in only 10-60 min for protein characterization with MS, the sample can be recovered from the MALDI target plate for use in ESI MS, and there was a 90% reduction in sample-processing time from overnight to ca. 3 h.     

Liquid chromatography and electron-capture dissociation in Fourier transform ion cyclotron resonance mass spectrometry

Liquid separation methods in combination with electrospray mass spectrometry as well as the recently introduced fragmentation method electron capture dissociation (ECD) have become powerful tools in proteomics research. This paper presents the results of the first successful attempts to combine liquid chromatography (LC) and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) with ECD in the analysis of a mixture of standard peptides and of a bovine serum albumin tryptic digest. A novel electron injection system provided conditions for ECD sufficient to yield extensive sequence information for the most abundant peptides in the mixtures on the time-scale of the chromatographic separation. The results suggest that LC/ECD-FTICRMS can be employed in the characterization of peptides in enzymatic digests of proteins or protein mixtures and identify and localize posttranslational modifications.     

Electrophoretic mobility-assisted identification of proteins by nanoelectrospray capillary electrophoresis/mass spectrometry under methanolic conditions

A novel method for electrophoretic mobility-assisted identifications of proteins, using capillary electrophoresis/mass spectrometry (CE/MS) under methanolic conditions, was developed. The number of functional groups of the enzymatic digest peptides was estimated from a single run CE/MS analysis and utilized as an additional tag for database searching in addition to the mass map of the peptides. The additional amino acid information thus obtained can improve the confidence level of the protein identification. The database searching software algorithm ProFound was modified to accept the tag, based on this new concept. In this study, optimization of the CE/MS conditions for the estimation of basic functional groups was performed as an example. An accurate value of the number of such functional groups was obtained from CE characteristics when methanolic buffer (methanol/formic acid/water = 60:20:20) was used, via an excellent correlation (r = 0.997) between the number of functional groups of the peptides and [MW((2/3))]. The mass spectrometry sensitivity was also improved when using the methanolic buffer in comparison with that obtained using aqueous 1% formic acid buffer. The identification of a protein of Saccharomyces cerevisiae, which was separated by two-dimensional electrophoresis, was performed using the methanolic buffer in combination with sheathless nanoelectrospray CE/MS. A protein spot that had not been identified by MALDI-TOFMS and LC/MS/MS was successfully identified using this new method.     

Capillary electrophoresis/tandem mass spectrometry for analysis of proteins from two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis

Capillary electrophoresis/time-of-flight mass spectrometry(CE/TOFMS) has been used for analysis of in-gel digests of protein spots excised from two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE). An off-line purification and preconcentration procedure with a Zip Tip is used before CE/TOFMS analysis which allows for detection of protein spots with <1 picomole of material from 2-D gels. The off-line procedure provides sufficient purification for analysis while maintaining the quality of the CE separation. Using this procedure, several proteins from Coomassie Blue and zinc negatively stained gels are identified by the peptide maps generated and database searching. CE/TOF tandem mass spectrometry is used for the confirmation of database searching results and structural analysis of peptides that do not match the expected peptide maps obtained from the database in order to identify structural modifications. Several modifications were pinpointed and identified by this method.     

On-line capillary separations/tandem mass spectrometry for protein digest analysis by using an ion trap storage/reflectron time-of flight mass detector

Capillary separations interfaced to tandem mass spectrometry provide a very powerful tool for the characterization of biological macromolecules such as proteins and peptides. The development of real time data-dependent data acquisition has further enhanced the capability of this method. However, the application of this technique to fast capillary separations has been limited by the relatively slow spectral acquisition speed available on scanning mass spectrometers. In this work, an ion trap storage/reflectron time-of-flight mass spectrometer (IT/reTOF-MS) has been used as an on-line tandem mass detector for capillary high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) separations of peptide mixtures including a protein digest. By taking advantage of the nonscanning property of the time-of-flight mass spectrometer, a fast spectral acquisition rate has been achieved. This fast spectral acquisition rate, combined with a new protocol that speeds up tickle voltage optimization, has provided MS/MS spectra for multiple components in a hemoglobin digest during one liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) run. Further, the IT/reTOF-MS has the speed to provide MS/MS spectra for multiple components in a CE separation of a synthetic peptide mixture within one CE/MS/MS run.     

Tandem mass spectrometry methods for definitive protein identification in proteomics research

Optimized procedures have been developed for the addition of sulfonic acid groups to the N-termini of low-level peptides. These procedures have been applied to peptides produced by tryptic digestion of proteins that have been separated by two-dimensional (2-D) gel electrophoresis. The derivatized peptides were sequenced using matrix-assisted laser desorption/ionization (MALDI) post-source decay (PSD) and electrospray ionization-tandem mass spectrometry methods. Reliable PSD sequencing results have been obtained starting with sub-picomole quantities of protein. We estimate that the current PSD sequencing limit is about 300 fmol of protein in the gel. The PSD mass spectra of the derivatized peptides usually allow much more specific protein sequence database searches than those obtained without derivatization. We also report initial automated electrospray ionization-tandem mass spectrometry sequencing of these novel peptide derivatives. Both types of tandem mass spectra provide predictable fragmentation patterns for arginine-terminated peptides. The spectra are easily interpreted de novo, and they facilitate error-tolerant identification of proteins whose sequences have been entered into databases.     

Using HPTLC/DESI-MS for peptide identification in 1D separations of tryptic protein digests

Desorption electrospray ionization mass spectrometry (DESI-MS) was investigated as a method to detect and identify peptides from tryptic digests of cytochrome c and myoglobin separated on ProteoChrom HPTLC Silica gel 60 F(254s) plates and ProteoChrom HPTLC Cellulose sheets. Full-scan mass spectra and data-dependent tandem mass spectra were acquired in separate plate scans and used to identify peptide ions. Peptide distributions along the development lane were mapped for each separated protein digest. Signal levels ranged over several orders of magnitude. In general, highest signal levels were obtained for the peptides with the highest R (f) values on a plate, while peptides with very low R (f) values were often not detected. Sequence coverages for cytochrome c were 58% for the digest separated on the silica gel plate and 72% for the separation on the cellulose sheet; myoglobin sequence coverages were 62% and 68% on silica gel and cellulose, respectively. Weak correlations between peptide hydrophilicity and R (f) values on the silica gel and cellulose plates were found, with the more hydrophilic peptides having lower R (f) values.     

Extraction and characterization of adenovirus

A new methodology for the extraction and characterization of proteins from Coomassie-stained sodium dodecylsulfate polyacrylamide gel electrophoresis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been described. The utility of this methodology was demonstrated in the characterization of adenovirus proteins. The key steps in the extraction and destaining process involve washing the excised band with a combination of solvents that include 10% acetic acid, acetonitrile, methanol, and formic acid:water:isopropanol mixture. By using this procedure, we determined adenovirus proteins with molecular weights ranging from 10,000 to 110,000 Da by MALDI-MS, obtaining a detection limit of approximately 6 pmol. Parallel experiments were successfully carried out to analyze adenovirus proteins from Cu-stained gels. It was observed that increase in laser intensity resulted in significant improvements in the quality of MALDI mass spectra for the analysis of inefficiently destained proteins from Cu-stained gels.     

Study of Burkitt lymphoma cell line proteins by high resolution two-dimensional gel electrophoresis and nanoelectrospray mass spectrometry

We paper describe a mass spectrometric approach generally applicable for the rapid identification and characterization of proteins isolated by two-dimensional gel electrophoresis (2-DE). The highly sensitive nanoflow-electrospray mass spectrometry employing a quadrupole-time of flight mass spectrometer was used for the direct identification of proteins from the peptide mixture generated from only one high resolution 2-DE gel without high performance liquid chromatography (HPLC) separation or Edman sequencing. Due to the high sensitivity and high mass accuracy of the instrument employed, this technique proved to be a powerful tool for the identification of proteins from femtomole amounts of materials. We applied the technique for the investigation of Burkitt lymphoma BL60 cell proteins. This cell line has been used as a model to assign apoptosis-associated proteins by subtractive analysis of normal and apoptotic cells. From the nuclear fraction of these cells, 36 protein spots were examined, from only one micropreparative Coomassie Brilliant Blue R-250 stained gel, after proteolytic digestion by matrix assisted laser desorption ionization (MALDI) and nanospray mass spectrometry (MS). In combination with database searches, of 33 proteins were successfully identified by nanospray-MS/MS-sequencing of up to eight peptides per protein. Three proteins were new proteins not listed in any of the available databases. Some of the identified proteins are known to be involved in apoptosis processes, the others were common proteins in the eukaryotic cell. The given technique and the protein data are the basis for construction of a database to compare normal and apoptosis-induced cells and, further, to enable fast screening of drug impact in apoptosis-associated processes.     

A simplified device for protein identification by microcapillary gradient liquid chromatography-tandem mass spectrometry

A simplified device and procedure have been developed for microcapillary gradient liquid chromatography-tandem mass spectrometry (LC-MS/MS). This procedure has proved useful in identifying low level quantities of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel bands. Microelectrospray needles are packed with reversed-phase resin and function both as a high performance liquid chromatography (HPLC) column and a nanospray mass spectrometer tip when interfaced between an HPLC and ion trap mass spectrometer. Variable submicroliter flow rates are generated by flow splitting between the microelectrospray capillary and an HPLC system. A manual injector is used to inject a protein digest mixture that binds to the column and is then washed at a high flow rate (2 microL/min post split). Gradient elution of bound peptides was initiated by the injection of a filled loop of 70% v/v methanol (5 microL) concomitant with a reduction of flow rate (0.1 microL/min post split). This forms a diffusion-dependent gradient of variable length (typically 15-30 min in length) depending upon the final flow rate. Chromatographic separations of a standard solution digest demonstrate that this diffusion-dependent gradient provides reasonable separations such that multiple peptide identifications by MS/MS can be obtained. Application of this methodology to the analysis of several in-gel-digested gel-separated proteins is presented to demonstrate its utility.     

Identification of platelet proteins separated by two-dimensional gel electrophoresis and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and detection of tyrosine-phosphorylated proteins

Different search programs were compared to judge their particular efficiency in protein identification. We established a human blood platelet protein map and identified tyrosine-phosphorylated proteins. The cytosolic fraction of human blood platelets was separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and phosphorylated proteins were detected by Western blotting using anti-phosphotyrosine antibodies. Visualized protein spots were excised, digested with trypsin and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The obtained mass fingerprint data sets have been analyzed using ProFound, MS-Fit and Mascot. For those protein spots with no significant search results MALDI post source decay (PSD) spectra have been acquired on the same sample. For automatic interpretation of these fragment ion spectra, the SEQUEST and Mascot algorithm were applied. Another approach for the identification of phosphorylated proteins is immunoprecipitation using an anti-phosphotyrosine antibody. A method for immunoprecipitation of tyrosine-phosphorylated peptides was optimized.     

An improved calibration method for the matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resononance analysis of 15N-metabolically- labeled proteome digests using a mass difference approach

High mass measurement accuracy of peptides in enzymatic digests is critical for confident protein identification and characterization in proteomics research. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) can provide low or sub-ppm mass accuracy and ultrahigh resolving power. While for ESI-FT-ICR-MS, the mass accuracy is generally 1 ppm or better, with matrix-assisted laser desorption/ionization (MALDI)-FT-ICR-MS, the mass errors can vary from sub-ppm with internal calibration to over 100 ppm with conventional external calibration. A novel calibration method for (15)N-metabolically labeled peptides from a batch digest of a proteome is described which corrects for space charge induced frequency shifts in FT-ICR spectra without using an internal calibrant. This strategy utilizes the information from the mass difference between the (14)N/(15)N peptide peak pairs to correct for space charge induced mass shifts after data collection. A procedure for performing the mass correction has been written into a computer program and has been successfully applied to high-performance liquid chromatography-MALDI-FT- ICR-MS measurement of (15)N-metabolic labeled proteomes. We have achieved an average measured mass error of 1.0 ppm and a standard deviation of 3.5 ppm for 900 peptides from 68 MALDI-FT-ICR mass spectra of the proteolytic digest of a proteome from Methanococcus maripaludis.