Comparison of electrokinetics-based multidimensional separations coupled with electrospray ionization-tandem mass spectrometry for characterization of human salivary proteins

The foundation for saliva-based diagnostics is the development of a complete catalog of secreted proteins detectable in saliva. Besides protein complexity, the greatest bioanalytical challenge facing comprehensive analysis of saliva samples is related to the large variation of protein relative abundances including the presence of high-abundance proteins such as amylases, mucins, proline-rich proteins (PRPs), and secretory IgA complex. Among a number of electrokinetic separation techniques, transient capillary isotachophoresis/capillary zone electrophoresis (CITP/CZE) specifically targets trace amounts of proteins and thus reduces the range of relative protein abundances for providing unparallel advantages toward the identification of low-abundance proteins. By employing a CITP/CZE-based multidimensional separation platform coupled with electrospray ionization-tandem mass spectrometry (ESI-tandem MS), a total of 6112 fully tryptic peptides are sequenced at a 1% false discovery rate (FDR), leading to the identification of 1479 distinct human SwissProt protein entries. By comparing with capillary isoelectric focusing (CIEF) as another electrokinetics-based stacking approach, CITP/CZE not only offers a broad field of application but also is less prone to protein/peptide precipitation during the analysis. The ultrahigh resolving power of CITP/CZE is evidenced by the large number of distinct peptide identifications measured from each CITP fraction together with the low peptide fraction overlapping among identified peptides. Furthermore, when evaluating the protein sequence coverage by the number of distinct peptides mapping to each protein identification, the CITP-based proteome technology similarly achieves the superior performance with 674 proteins (46%) having three or more distinct peptides, 288 (19%) having two distinct peptides, and 517 (35%) having a single distinct peptide.     

Exploring the precursor ion exclusion feature of liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry for improving protein identification in shotgun proteome analysis

In shotgun proteome analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS), not all coeluting peptides at a given retention time are subjected to MS/MS due to the limitation of spectral acquisition speed of a mass spectrometer. In this work, precursor ion exclusion (PIE) in an electrospray ionization (ESI) quadrupole time-of-flight (QTOF) mass spectrometer was explored as a means of mitigating the undersampling problem. This strategy is based on running replicates of the sample where the precursor ions detected in the initial run(s) are excluded for MS/MS in the subsequent run. Four PIE methods as well as running replicates without PIE were investigated and compared for their effectiveness in identifying peptides and proteins. In the analysis of an MCF-7 breast cancer cell lysate digest by three replicate 2 h gradient LC-ESI runs, the first PIE method used a list of precursor ions detected in the initial run(s) for exclusion and identified a total of 572 proteins from the three runs combined with an average of 3.59 peptides matched to a protein. The second PIE method involved in the generation of a list of m/ z values of precursor ions along with their retention time information from the initial run(s), followed by entering these ions with retention times into the ion exclusion program of the QTOF control software for exclusion at a predefined retention time window (i.e., +/-150 s). In comparison to the first PIE method, this method reduced the possibility of excluding different peptide ions of the same m/ z (within a mass tolerance window) eluted at different retention windows. A total of 657 proteins were identified with an average of 3.75 peptides matched to a protein. The third PIE method studied relied on the exclusion of the precursor ions of peptides identified through database search of the MS/MS spectra generated in the initial run(s). This selective PIE method identified a total of 681 proteins with an average of 3.68 peptides matched to a protein. The final PIE method investigated involves the expansion of the selective PIE list by including nonidentifiable peptide ions found in the database search. This complete PIE method identified a total of 726 proteins with an average of 3.66 peptides per protein. In the case of three replicate runs without PIE, a total of 460 proteins were identified with an average of 3.51 peptides matched to a protein. Thus, the use of an optimal PIE strategy significantly increased the number of proteins identified from replicate runs (i.e., 726 vs 460 or a 58% increase). It is further demonstrated that this PIE strategy also improves protein identification efficiency in the analysis of a yeast whole cell lysate digesta less complex proteome digest. A total of 533 proteins identified from five replicate runs with complete PIE, compared to 353 proteins identified from the five replicate runs without PIE, representing a 51% increase in the number of proteins identified.     

Separation and identification of peptides from gel-isolated membrane proteins using a microfabricated device for combined capillary electrophoresis/nanoelectrospray mass spectrometry

The coupling of microfabricated devices to nanoelectrospray mass spectrometers using both a triple quadrupole and a quadrupole time-of-flight mass spectrometer (QqTOF MS) is presented for the analysis of trace-level membrane proteins. Short disposable nanoelectrospray emitters were directly coupled to the chip device via a low dead volume connection. The analytical performance of this integrated device in terms of sensitivity and reproducibility was evaluated for standard peptide mixtures. A concentration detection limit ranging from 3.2 to 43.5 nM for different peptides was achieved in selected ion monitoring, thus representing a 10-fold improvement in sensitivity compared to that of microelectrospray using the same chip/mass spectrometer. Replicate injections indicated that reproducibility of migration time was typically less than 3.1% RSD whereas RSD values of 6-13% were observed on peak areas. Although complete resolution of individual components is not typically achieved for complex digests, the present chip capillary electrophoresis (chip-CE) device enabled proper sample cleanup and partial separation of multicomponent samples prior to mass spectral identification. Analyses of protein digests were typically achieved in less than 1.5 min with peak widths of 1.8-2.5 s (half-height definition) as indicated from individual reconstructed ion electropherograms. The application of this chip-CE/QqTOF MS system is further demonstrated for the identification of membrane proteins which form a subset of the Haemophilus influenzae proteome. Bands first separated by 1D-gel electrophoresis were excised and digested, and extracted tryptic peptides were loaded on the chip without any further sample cleanup or on-line adsorption preconcentration. Accurate molecular mass determination (< 5 ppm) in peptide-mapping experiments was obtained by introducing an internal standard via a postseparation channel. The analytical potential of this integrated device for the identification of trace-level proteins from different strains of H. influenzae is demonstrated using both peptide mass-fingerprint database searching and on-line tandem mass spectrometry.     

Proteome-wide identification of proteins and their modifications with decreased ambiguities and improved false discovery rates using unique sequence tags

Identifying proteins and their modification states and with known levels of confidence remains as a significant challenge for proteomics. Random or decoy peptide databases are increasingly being used to estimate the false discovery rate (FDR), e.g., from liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of tryptic digests. We show that this approach can significantly underestimate the FDR and describe an approach for more confident protein identifications that uses unique partial sequences derived from a combination of database searching and amino acid residue sequencing using high-accuracy MS/MS data. Applied to a Saccharomyces cerevisiae tryptic digest, the approach provided 3 132 confident peptide identifications ( approximately 5% modified in some fashion), covering 575 proteins with an estimated zero FDR. The conventional approach provided 3 359 peptide identifications and 656 proteins with 0.3% FDR based upon a decoy database analysis. However, the present approach revealed approximately 5% of the 3 359 identifications to be incorrect and many more as potentially ambiguous (e.g., due to not considering certain amino acid substitutions and modifications). In addition, 677 peptides and 39 proteins were identified that had been missed by conventional analysis, including nontryptic peptides, peptides with a variety of expected/unexpected chemical modifications, known/unknown post-translational modifications, single nucleotide polymorphisms or gene encoding errors, and multiple modifications of individual peptides.     

Integration of electrokinetic-based multidimensional separation/concentration platform with electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry for proteome analysis of Shewanella oneidensis

This work focuses on the development of a multidimensional electrokinetic-based separation/concentration platform coupled with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) for achieving the high resolution and ultrasensitive analysis of complex protein/peptide mixtures. A microdialysis junction is employed as the interface for on-line combination of capillary isoelectric focusing (CIEF) with transient capillary isotachophoresis/zone electrophoresis (CITP/CZE) in an integrated platform. Besides the excellent resolving power afforded by both CIEF and CZE separations, the electrokinetic focusing/stacking effects of CIEF and CITP greatly enhance the dynamic range and detection sensitivity of MS for protein identification. The constructed multidimensional separation/concentration platform is demonstrated for the analysis of Shewanella oneidensis proteome, which has considerable implications toward the bioremediation of environmental pollutants. The electrokinetic-based platform offers the overall peak capacity comparable to those obtained using multidimensional chromatography systems, but with a much shorter run time and no need for column regeneration. Most importantly, a total of 1174 unique proteins, corresponding to 26.5% proteome coverage, are identified from the cytosolic fraction of S. oneidensis, while requiring <500 ng of proteolytic digest loaded in the CIEF capillary. The ultrasensitive capabilities of electrokinetic-based proteome approach are attributed to the concentration effect in CIEF, the electrokinetic stacking of CITP, the nanoscale peak volume in CZE, the "accurate mass tag" strategy for protein/peptide identification, and the high-sensitivity, high-resolution, and high-mass measurement accuracy of FTICR-MS.     

Dynamic range expansion applied to mass spectrometry based on data-dependent selective ion ejection in capillary liquid chromatography fourier transform ion cyclotron resonance for enhanced proteome characterization.

The characterization of cellular proteomes is important for understanding biochemical processes ranging from cell differentiation to cancer development. In one highly promising approach, whole protein extracts or fractions are digested (e.g., with trypsin) and injected into a packed capillary column for subsequent separation. The separated peptides are then introduced on-line to an electrospray ionization source of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer for the detection of peptide accurate mass tags that serve as biomarkers for their parent proteins. In this work, we report the use of data-dependent selective external ion ejection in conjunction with FTICR and on-line capillary LC separations for the enhanced characterization of peptide mixtures and a yeast extract proteome. The number of peptides identified in an LC-FTICR analysis of a yeast proteome digest employing data-dependent rf-only dipolar ejection of the most abundant ion species prior to ion accumulation was 40% higher than that detected in a separate LC-FTICR analysis using conventional nonselective ion accumulation.     

Enhanced characterization of complex proteomic samples using LC-MALDI MS/MS: exclusion of redundant peptides from MS/MS analysis in replicate runs

Due to the complexity of proteome samples, only a portion of peptides and thus proteins can be identified in a single LC-MS/MS analysis in current shotgun proteomics methodologies. It has been shown that replicate runs can be used to improve the comprehensiveness of the proteome analysis; however, high-intensity peptides tend to be analyzed repeatedly in different runs, thus reducing the chance of identifying low-intensity peptides. In contrast to commonly used online ESI-MS, offline MALDI decouples the separation from MS acquisition, thus allowing in-depth selection for specific precursor ions. Accordingly, we extended a strategy for offline LC-MALDI MS/MS analysis using a precursor ion exclusion list consisting of all identified peptides in preceding runs. The exclusion list eliminated redundant MS/MS acquisitions in subsequent runs, thus reducing MALDI sample depletion and allowing identification of a larger number of peptide identifications in the cumulative dataset. In the analysis of the digest of an Escherichia coli lysate, the exclusion list strategy resulted in a 25% increase in the number of unique peptide identifications in the second run, in contrast to simply pooling MS/MS data from two replicate runs. To reduce the increased LC analysis time for repeat runs, a four-column multiplexed LC system was developed to carry out separation simultaneously. The multiplexed LC-MALDI MS provides a high-throughput platform to utilize the exclusion list strategy in proteome analysis.     

Analysis of the acidic proteome with negative electron-transfer dissociation mass spectrometry

We describe the first implementation of negative electron-transfer dissociation (NETD) on a hybrid ion trap-orbitrap mass spectrometer and its application to high-throughput sequencing of peptide anions. NETD, coupled with high pH separations, negative electrospray ionization (ESI), and an NETD compatible version of OMSSA, is part of a complete workflow that includes the formation, interrogation, and sequencing of peptide anions. Together these interlocking pieces facilitated the identification of more than 2000 unique peptides from Saccharomyces cerevisiae representing the most comprehensive analysis of peptide anions by tandem mass spectrometry to date. The same S. cerevisiae samples were interrogated using traditional, positive modes of peptide LC-MS/MS analysis (e.g., acidic LC separations, positive ESI, and collision activated dissociation), and the resulting peptide identifications of the different workflows were compared. Due to a decreased flux of peptide anions and a tendency to produce lowly charged precursors, the NETD-based LC-MS/MS workflow was not as sensitive as the positive mode methods. However, the use of NETD readily permits access to underrepresented acidic portions of the proteome by identifying peptides that tend to have lower pI values. As such, NETD improves sequence coverage, filling out the acidic portions of proteins that are often overlooked by the other methods.     

Robust analysis of the yeast proteome under 50 kDa by molecular-mass-based fractionation and top-down mass spectrometry

As the process of top-down mass spectrometry continues to mature, we benchmark the next installment of an improving methodology that incorporates a tube-gel electrophoresis (TGE) device to separate intact proteins by molecular mass. Top-down proteomics is accomplished in a robust fashion to yield the identification of hundreds of unique proteins, many of which correspond to multiple protein forms. The TGE platform separates 0-50 kDa proteins extracted from the yeast proteome into 12 fractions prior to automated nanocapillary LC-MS/MS in technical triplicate. The process may be completed in less than 72 h. From this study, 530 unique proteins and 1103 distinct protein species were identified and characterized, thus representing the highest coverage to date of the Saccharomyces cerevisiae proteome using top-down proteomics. The work signifies a significant step in the maturation of proteomics based on direct measurement and fragmentation of intact proteins.     

Tandem parallel fragmentation of peptides for mass spectrometry

Parallel fragmentations of peptides in the source region and in the collision cell of tandem mass spectrometers are sequentially combined to develop parallel collision-induced-dissociation mass spectrometry (p2CID MS). Compared to MS/MS spectra, the p2CID mass spectra show increased signal intensities (2-400-fold) and number of sequence ions. This improvement is attributed to the fact that p2CID MS virtually samples all the ions generated by electrospray ionization, including intact and fragment ions of different charge states from a peptide. We implement the method using a quadrupole time-of-flight tandem mass spectrometer. The instrument is operated in TOF-MS mode that allows the ions from source region broadband-passing the first mass analyzer to enter the collision cell. Cone voltage and collision energy are investigated to optimize the outcome of the two parallel CID processes. In the in-source parallel CID, elevated cone voltage produces singly charged intact peptide ions and large fragment ions, as well as decreases the charge-state distribution of peptide ions mainly to double and single charges. The in-collision-cell parallel CID is optimized to dissociate the ions from the source region to produce small and medium fragment ions. The method of p2CID MS is especially useful for sequencing of large peptides with labile amide bonds and peptides with C-terminal arginine. It has unique potential for de novo sequencing of peptides and proteome analysis, especially for affinity-enriched subproteomes.     

On-line capillary electrophoresis/microelectrospray ionization-tandem mass spectrometry using an ion trap storage/time-of-flight mass spectrometer with SWIFT technology.

The development of a system capable of the speed required for on-line capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) of tryptic digests is described. The ion trap storage/reflectron time-of-flight (IT/reTOF) mass spectrometer is used as a nonscanning detector for rapid CE separation, where the peptides are ionized on-line using electrospray ionization (ESI). The ESI produced ions are stored in the ion trap and dc pulse injected into the reTOF-MS at a rate sufficient to maintain the separation achieved by CE. Using methodology generated by software and hardware developed in our lab, we can produce SWIFT (Stored Waveform Inverse Fourier Transform) ion isolation and TICKLE activation/fragmentation voltage waveforms to generate MS/MS at a rate as high as 10 Hz so that the MS/MS spectra can be optimized on even a 1-2 s eluting peak. In CE separations performed on tryptic digests of dogfish myelin basic protein (MBP) where eluting peaks 4-8 s wide are observed, it is demonstrated that an acquisition rate of 4 Hz provides > 20 spectra/peak and is more than sufficient to provide optimized MS/MS spectra of each of the eluting peaks in the electropherogram. The detailed structural analysis of dogfish MBP including several posttranslational modifications using CE-MS and CE-MS/MS is demonstrated using this method with < 10 fmol of material consumed.     

Direct profiling of proteins in biological tissue sections by MALDI mass spectrometry

The direct profiling of proteins present in tissue sections for several organs of the mouse has been accomplished using matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). Fresh tissue was sectioned and blotted on a conductive polyethylene membrane. The dried membrane blot was coated with matrix, typically sinapinic acid, and directly analyzed in the mass spectrometer. Generally, well over 100 peptide/protein signals in the 2000-30,000 Da range were observed, with 30-50 having relatively high signal intensities. Analysis of different areas of the same tissue gave remarkably similar mass spectra with greater than 90% homology. However, different parts of a segmented tissue, such as the proximal, intermediate, and distal colon, gave some unique protein signals. After treatment of the tissue blot with protease and subsequent MALDI MS analysis using postsource decay methods for peptide sequencing, some of the proteins were identified. The unique protein profiles measured from these tissue blots also showed differences from strain to strain of the mouse, with genetically similar strains having very similar patterns.     

Online nanoflow reversed phase-strong anion exchange-reversed phase liquid chromatography-tandem mass spectrometry platform for efficient and in-depth proteome sequence analysis of complex organisms

The dynamic range of protein expression in complex organisms coupled with the stochastic nature of discovery-driven tandem mass spectrometry (MS/MS) analysis continues to impede comprehensive sequence analysis and often provides only limited information for low-abundance proteins. High-performance fractionation of proteins or peptides prior to mass spectrometry analysis can mitigate these effects, though achieving an optimal combination of automation, reproducibility, separation peak capacity, and sample yield remains a significant challenge. Here we demonstrate an automated nanoflow 3-D liquid chromatography (LC)-MS/MS platform based on high-pH reversed phase (RP), strong anion exchange (SAX), and low-pH reversed phase (RP) separation stages for analysis of complex proteomes. We observed that RP-SAX-RP outperformed RP-RP for analysis of tryptic peptides derived from Escherichia coli and enabled identification of proteins present at a level of 50 copies per cell in Saccharomyces cerevisiae, corresponding to an estimated detection limit of 500 amol, from 40 μg of total lysate on a low-resolution 3-D ion trap mass spectrometer. A similar study performed on a LTQ-Orbitrap yielded over 4000 unique proteins from 5 μg of total yeast lysate analyzed in a single, 101 fraction RP-SAX-RP LC-MS/MS acquisition, providing an estimated detection limit of 65 amol for proteins expressed at 50 copies per cell.     

Determination of bioactive peptides using capillary zone electrophoresis/mass spectrometry.

Mixtures of bioactive peptides have been analyzed by capillary zone electrophoresis/mass spectrometry (CZE/MS) using an on-line coaxial continuous-flow fast atom bombardment interface. High separation efficiencies (up to 410,000 theoretical plates) were obtained from low femtomole levels of peptides. The analysis of basic peptides was accomplished by using aminopropyl-silylated CZE columns to minimize zone broadening due to adsorption effects. CZE/MS/MS data were acquired from femtomole levels of peptides in electrophoretic real time.     

Targeted comparative proteomics by liquid chromatography-tandem Fourier ion cyclotron resonance mass spectrometry

In proteomics, effective methods are needed for identifying the relatively limited subset of proteins displaying significant changes in abundance between two samples. One way to accomplish this task is to target for identification by MS/MS only the "interesting" proteins based on the abundance ratio of isotopically labeled pairs of peptides. We have developed the software and hardware tools for online LC-FTICR MS/MS studies in which a set of initially unidentified peptides from a proteome analysis can be selected for identification based on their distinctive changes in abundance following a "perturbation". We report here the validation of this method using a mixture of standard proteins combined in different ratios after isotopic labeling. We also demonstrate the application of this method to the identification of Shewanella oneidensis peptides/proteins exhibiting differential abundance in suboxic versus aerobic cell cultures.     

Fully integrated glass microfluidic device for performing high-efficiency capillary electrophoresis and electrospray ionization mass spectrometry

A microfabricated device has been developed in which electrospray ionization is performed directly from the corner of a rectangular glass microchip. The device allows highly efficient electrokinetically driven separations to be coupled directly to a mass spectrometer (MS) without the use of external pressure sources or the insertion of capillary spray tips. An electrokinetic-based hydraulic pump is integrated on the chip that directs eluting materials to the monolithically integrated spray tip. A positively charged surface coating, PolyE-323, is used to prevent surface interactions with peptides and proteins and to reverse the electroosmotic flow in the separation channel. The device has been used to perform microchip CE-MS analysis of peptides and proteins with efficiencies over 200,000 theoretical plates (1,000,000 plates/m). The sensitivity and stability of the microfabricated ESI source were found to be comparable to that of commercial pulled fused-silica capillary nanospray sources.     

Quantification of intermediate-abundance proteins in serum by multiple reaction monitoring mass spectrometry in a single-quadrupole ion trap

A method is presented to quantify intermediate-abundance proteins in human serum using a single-quadrupole linear ion trap mass spectrometer-in contrast, for example, to a triple-quadrupole mass spectrometer. Stable-isotope-labeled (tryptic) peptides are spiked into digested protein samples as internal standards, aligned with the traditional isotope dilution approach. As a proof-of-concept experiment, four proteins of intermediate abundance were selected, coagulation factor V, adiponectin, C-reactive protein (CRP), and thyroxine binding globulin. Stable-isotope-labeled peptides were synthesized with one tryptic sequence from each of these proteins. The normal human serum concentration ranges of these proteins are from 1 to 30 microg/mL (or 20 to 650 pmol/mL). These labeled peptides and their endogenous counterparts were analyzed by LC-MS/MS using multiple reaction monitoring, a multiplexed form of the selected reaction monitoring technique. For these experiments, only one chromatographic dimension (on-line reversed-phase capillary column) was used. Improved limits of detection will result with multidimensional chromatographic methods utilizing more material per sample. Standard curves of the spiked calibrants were generated with concentrations ranging from 3 to 700 pmol/mL using both neat solutions and peptides spiked into the complex matrix of digested serum protein solution where ion suppression effects and interferences are common. Endogenous protein concentrations were determined by comparing MS/MS peak areas of the endogenous peptides to the isotopically labeled internal calibrants. The derived concentrations from a normal human serum pool (neglecting loss of material during sample processing) were 9.2, 110, 120, and 246 pmol/mL for coagulation factor V, adiponectin, CRP, and thyroxine binding globulin, respectively. These concentrations generally agree with the reported normal ranges for these proteins. As a measure of analytical reproducibility of this single-quadrupole assay, the coefficients of variance based on 12 repeated measurements for each of the endogenous tryptic peptides were 17.0, 25.4, 24.2, and 14.0% for coagulation factor V, adiponectin, CRP, and thyroxine binding globulin, respectively.     

Processing complex mixtures of intact proteins for direct analysis by mass spectrometry

For analysis of intact proteins by mass spectrometry (MS), a new twist to a two-dimensional approach to proteome fractionation employs an acid-labile detergent instead of sodium dodecyl sulfate during continuous-elution gel electrophoresis. Use of this acid-labile surfactant (ALS) facilitates subsequent reversed-phase liquid chromatography (RPLC) for a net two-dimensional fractionation illustrated by transforming thousands of intact proteins from Saccharomyces cerevisiae to mixtures of 5-20 components (all within approximately 5 kDa of one another) for presentation via electrospray ionization (ESI) to a Fourier transform MS (FTMS). Between 3 and 13 proteins have been detected directly using ESI-FTMS (or MALDI-TOF), and the fractionation showed a peak capacity of approximately 400 between 0 and 70 kDa. A probability-based identification was made automatically from raw MS/MS data (obtained using a quadrupole-FTMS hybrid instrument) for one protein that differed from that predicted in a yeast database of approximately 19,000 protein forms. This ALS-PAGE/RPLC approach to proteome processing ameliorates the "front end" problem that accompanies direct analysis of whole proteins and assists the future realization of protein identification with 100% sequence coverage in a high-throughput format.     

High-throughput peptide identification from protein digests using data-dependent multiplexed tandem FTICR mass spectrometry coupled with capillary liquid chromatography

Tandem mass spectrometry (MS/MS) plays an important role in the unambiguous identification and structural elucidation of biomolecules. In contrast to conventional MS/MS approaches for protein identification where an individual polypeptide is sequentially selected and dissociated, a multiplexed-MS/MS approach increases throughput by selecting several peptides for simultaneous dissociation using either infrared multiphoton dissociation (IRMPD) or multiple frequency sustained off-resonance irradiation (SORI) collisionally induced dissociation (CID). The high mass measurement accuracy and resolution of FTICR combined with knowledge of peptide dissociation pathways allows the fragments arising from several different parent ions to be assigned. Herein we report the application of multiplexed-MS/MS coupled with on-line separations for the identification of peptides present in complex mixtures (i.e., whole cell lysate digests). Software was developed to enable "on-the-fly" data-dependent peak selection of a subset of polypeptides from each FTICR MS acquisition. In the subsequent MS/MS acquisitions, several coeluting peptides were fragmented simultaneously using either IRMPD or SORI-CID techniques. The utility of this approach has been demonstrated using a bovine serum albumin tryptic digest separated by capillary LC where multiple peptides were readily identified in single MS/MS acquisitions. We also present initial results from multiplexed-MS/MS analysis of a D. radiodurans whole cell digest to illustrate the utility of this approach for high-throughput analysis of a bacterial proteome.     

Identification of bacteria using tandem mass spectrometry combined with a proteome database and statistical scoring

Detection and identification of pathogenic bacteria and their protein toxins play a crucial role in a proper response to natural or terrorist-caused outbreaks of infectious diseases. The recent availability of whole genome sequences of priority bacterial pathogens opens new diagnostic possibilities for identification of bacteria by retrieving their genomic or proteomic information. We describe a method for identification of bacteria based on tandem mass spectrometric (MS/MS) analysis of peptides derived from bacterial proteins. This method involves bacterial cell protein extraction, trypsin digestion, liquid chromatography MS/MS analysis of the resulting peptides, and a statistical scoring algorithm to rank MS/MS spectral matching results for bacterial identification. To facilitate spectral data searching, a proteome database was constructed by translating genomes of bacteria of interest with fully or partially determined sequences. In this work, a prototype database was constructed by the automated analysis of 87 publicly available, fully sequenced bacterial genomes with the GLIMMER gene finding software. MS/MS peptide spectral matching for peptide sequence assignment against this proteome database was done by SEQUEST. To gauge the relative significance of the SEQUEST-generated matching parameters for correct peptide assignment, discriminant function (DF) analysis of these parameters was applied and DF scores were used to calculate probabilities of correct MS/MS spectra assignment to peptide sequences in the database. The peptides with DF scores exceeding a threshold value determined by the probability of correct peptide assignment were accepted and matched to the bacterial proteomes represented in the database. Sequence filtering or removal of degenerate peptides matched with multiple bacteria was then performed to further improve identification. It is demonstrated that using a preset criterion with known distributions of discriminant function scores and probabilities of correct peptide sequence assignments, a test bacterium within the 87 database microorganisms can be unambiguously identified.