Two-dimensional method for time aligning liquid chromatography-mass spectrometry data

We describe a new time alignment method that takes advantage of both dimensions of LC-MS data to resolve ambiguities in peak matching while remaining computationally efficient. This approach, Warp2D, combines peak extraction with a two-dimensional correlation function to provide a reliable alignment scoring function that is insensitive to spurious peaks and background noise. One-dimensional alignment methods are often based on the total-ion-current elution profile of the spectrum and are unable to distinguish peaks of different masses. Our approach uses one-dimensional alignment in time, but with a scoring function derived from the overlap of peaks in two dimensions, thereby combining the specificity of two-dimensional methods with the computational performance of one-dimensional methods. The peaks are approximated as two-dimensional Gaussians of varying width. This approximation allows peak overlap (the measure of alignment quality) to be calculated analytically, without computationally intensive numerical integration in two dimensions. To demonstrate the general applicability of Warp2D, we chose a variety of complex samples that have substantial biological and analytical variability, including human serum and urine. We show that Warp2D works well with these diverse sample sets and with minimal tuning of parameters, based on the reduced standard deviation of peak elution times after warping. The combination of high computational speed, robustness with complex samples, and lack of need for detailed tuning makes this alignment method well suited to high-throughput LC-MS studies.     

Chromatographic alignment of ESI-LC-MS proteomics data sets by ordered bijective interpolated warping

Mass spectrometry proteomics typically relies upon analyzing outcomes of single analyses; however, comparing raw data across multiple experiments should enhance both peptide/protein identification and quantitation. In the absence of convincing tandem MS identifications, comparing peptide quantities between experiments (or fractions) requires the chromatographic alignment of MS signals. An extension of dynamic time warping (DTW), termed ordered bijective interpolated warping (OBI-Warp), is presented and used to align a variety of electrospray ionization liquid chromatography mass spectrometry (ESI-LC-MS) proteomics data sets. An algorithm to produce a bijective (one-to-one) function from DTW output is coupled with piecewise cubic hermite interpolation to produce a smooth warping function. Data sets were chosen to represent a broad selection of ESI-LC-MS alignment cases. High confidence, overlapping tandem mass spectra are used as standards to optimize and compare alignment parameters. We determine that Pearson's correlation coefficient as a measure of spectra similarity outperforms covariance, dot product, and Euclidean distance in its ability to produce correct alignments with optimal and suboptimal alignment parameters. We demonstrate the importance of penalizing gaps for best alignments. Using optimized parameters, we show that OBI-Warp produces alignments consistent with time standards across these data sets. The source and executables are released under MIT style license at http://obi-warp.sourceforge.net/.     

Large scale pesticide multiresidue methods in food combining liquid chromatography--time-of-flight mass spectrometry and tandem mass spectrometry

Liquid chromatography tandem mass spectrometry (LC-MS/MS) and liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) are powerful and complementary techniques that can independently cover the majority of the challenges related with pesticide residue food control. The sequential combination of both systems benefits from their complementary advantages and assists to increase the performance and to simplify routine large scale pesticide multiresidue methods. The proposed approach consists of three stages: (1) automated pesticide screening by LC-TOFMS; (2) identification by LC-TOFMS accurate mass measurements; and (3) confirmation and quantitation by LC-MS/MS. We have developed a fast comprehensive (identification/confirmation + quantitation) automated screening method for 100 target pesticides in crops. In the first stage, a set of data including m/z accurate mass windows (within 20 mDa width) and retention time is obtained (using a standard solution containing all the targeted pesticides) in order to build the automated screening procedure, which is created automatically by assigning retention time and the m/z mass window for each target pesticide. Samples are then analyzed, and the method enables the screening and preliminary identification of the species first by retention time and m/z mass window, followed by subsequent identification (only if positive results) by LC-TOFMS accurate mass measurements. After that, final confirmation of the positive findings using two MRM transitions and accurate quantitation is performed by LC-MS/MS using a hybrid triple quadrupole linear ion trap (QqLIT) mass spectrometer. In addition, the use of this QqLIT instrument also offers additional advantageous scanning modes (enhanced product ion and MS3 modes) for confirmatory purposes in compounds with poor fragmentation. Examples of applications to real samples show the potential of the proposed approach, including the detection of nonselected "a priori" compounds as a typical case of retrospective evaluation of banned or misused substances.     

High-throughput identification of proteins and unanticipated sequence modifications using a mass-based alignment algorithm for MS/MS de novo sequencing results

With the increasing availability of de novo sequencing algorithms for interpreting high-mass accuracy tandem mass spectrometry (MS/MS) data, there is a growing need for programs that accurately identify proteins from de novo sequencing results. De novo sequences derived from tandem mass spectra of peptides often contain ambiguous regions where the exact amino acid order cannot be determined. One problem this poses for sequence alignment algorithms is the difficulty in distinguishing discrepancies due to de novo sequencing errors from actual genomic sequence variation and posttranslational modifications. We present a novel, mass-based approach to sequence alignment, implemented as a program called OpenSea, to resolve these problems. In this approach, de novo and database sequences are interpreted as masses of residues, and the masses, rather than the amino acid codes, are compared. To provide further flexibility, the masses can be aligned in groups, which can resolve many de novo sequencing errors. The performance of OpenSea was tested with three types of data: a mixture of known proteins, a mixture of unknown proteins that commonly contain sequence variations, and a mixture of posttranslationally modified known proteins. In all three cases, we demonstrate that OpenSea can identify more peptides and proteins than commonly used database-searching programs (SEQUEST and ProteinLynx) while accurately locating sequence variation sites and unanticipated posttranslational modifications in a high-throughput environment.     

Top-down proteomics on a chromatographic time scale using linear ion trap fourier transform hybrid mass spectrometers

Proteomics has grown significantly with the aid of new technologies that consistently are becoming more streamlined. While processing of proteins from a whole cell lysate is typically done in a bottom-up fashion utilizing MS/MS of peptides from enzymatically digested proteins, top-down proteomics is becoming a viable alternative that until recently has been limited largely to offline analysis by tandem mass spectrometry. Here we describe a method for high-resolution tandem mass spectrometery of intact proteins on a chromatographic time scale. In a single liquid chromatography-tandem mass spectrometry (LC-MS/MS) run, we have identified 22 yeast proteins with molecular weights from 14 to 35 kDa. Using anion exchange chromatography to fractionate a whole cell lysate before online LC-MS/MS, we have detected 231 metabolically labeled (14N/15N) protein pairs from Saccharomyces cerevisiae. Thirty-nine additional proteins were identified and characterized from LC-MS/MS of selected anion exchange fractions. Automated localization of multiple acetylations on Histone H4 was also accomplished on an LC time scale from a complex protein mixture. To our knowledge, this is the first demonstration of top-down proteomics (i.e., many identifications) on linear ion trap Fourier transform (LTQ FT) systems using high-resolution MS/MS data obtained on a chromatographic time scale.     

Mass profile monitoring in trace analysis by gas chromatography/mass spectrometry.

The advantages of gas chromatography/mass spectrometry (GC/MS) selected-ion monitoring (SIM) in the mass profile (MP) mode at medium mass resolving power were investigated for analyses requiring detection of low-picogram quantities of analytes in complex mixtures. The mass profile monitoring provides a certainty at least 10 times greater than that achieved by conventional GC/MS-SIM in the peak-top monitoring mode, and it can be operated at lower mass resolving power to compensate for the loss of sensitivity in the MP mode. The examination of mass profile peak shape, central mass shift, and sequential changes during GC elution not only reveals the presence of interfering compounds but also results in accurate mass measurement for those interferences. The latter feature takes the MP mode beyond the target mass analysis that GC/MS-SIM was originally designed for. This additional dimension of information is particularly useful for those complex and incompletely characterized matrices that are frequently encountered in environmental and biological sample analyses.     

Making broad proteome protein measurements in 1-5 min using high-speed RPLC separations and high-accuracy mass measurements

The throughput of proteomics measurements that provide broad protein coverage is limited by the quality and speed of both the separations as well as the subsequent mass spectrometric analysis; at present, analysis times can range anywhere from hours (high throughput) to days or longer (low throughput). We have explored the basis for proteomics analyses conducted on the order of minutes using high-speed capillary RPLC combined through on-line electrospray ionization interface with high-accuracy mass spectrometry (MS) measurements. Short 0.8-microm porous C18 particle-packed 50-microm-i.d. capillaries were used to speed the RPLC separations while still providing high-quality separations. Both time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FTICR) MS were applied for identifying peptides using the accurate mass and time (AMT) tag approach. Peptide RPLC relative retention (elution) times that were generated by solvent gradients that differed by at least 25-fold were found to provide relative elution times that agreed to within 5%, which provides the basis for using peptide AMT tags for higher throughput proteomics measurements. For fast MS acquisition speeds (e.g., 0.2 s for TOF and either approximately 0.3 or approximately 0.6 s for FTICR), peptide mass measurement accuracies of better than +/-15 ppm were obtained with the high-speed RPLC separations. The ability to identify peptides and the overall proteome coverage was determined by factors that include the separation peak capacity, the sensitivity of the MS (with fast scanning), and the accuracy of both the mass measurements and the relative RPLC peptide elution times. The experimental RPLC relative elution time accuracies of 5% (using high-speed capillary RPLC) and mass measurement accuracies of better than +/-15 ppm allowed for the confident identification of >2800 peptides and >760 proteins from >13,000 different putative peptides detected from a Shewanellaoneidensis tryptic digest. Initial results for both RPLC-ESI-TOF and RPLC-ESI-FTICR MS were similar, with approximately 2000 different peptides from approximately 600 different proteins identified within 2-3 min. For <120-s proteomic analysis, TOF MS analyses were more effective, while FTICR MS was more effective for the >150-s analysis due to the improved mass accuracies attained using longer spectrum acquisition times.     

Single-neuron analysis using CE combined with MALDI MS and radionuclide detection

Capillary electrophoresis (CE) has been combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and radionuclide detection to assay mass-limited biological samples. Nanovial sampling techniques enable injections into the CE capillary from 50 to 150-nL volume samples; after the separation, nanoliter fraction collection combines the CE effluent with a MALDI matrix and minimizes sample spreading, thus allowing both MALDI MS and radionuclide detection on the CE fractions. MALDI MS complements the elution time information of CE by providing accurate molecular mass data, and radionuclide detection provides zeptomole limits of detection with quantitative information. While MALDI MS detects all fully processed peptides at sufficient concentration, culturing the neuron in media containing 35S-Met provides selective radionuclide detection of newly synthesized methionine-containing peptides. The analysis and detection of the expected neuropeptides and hormones in a single 40-microm bag cell neuron from Aplysia californica with CE/MALDI MS/radionuclide detection demonstrates the ability of this hyphenated approach to work with chemically complex mass-limited samples.     

Automation of nanoscale microcapillary liquid chromatography-tandem mass spectrometry with a vented column

To fully automate the sample introduction step for nanoscale microcapillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses, 75 microm i.d. x 14 cm capillary columns were interfaced with a commercial autosampler instrument using a novel procedure which allowed dilute peptide samples to be transferred from the AS loop injector to the nanoscale column at flow rates up to 5 microL min(-1). On-column enrichment and desalting was demonstrated for large sample volumes (>40 microL) by constructing a vent 2 cm after the entrance to the packed bed of 5-microm ODS-AQ modified silica. Salts and nonretained solutes were removed via the vent, which allowed for column washing independent of the continuation of the bed into the electrospray source. Separations of test peptide mixtures demonstrated 50-nL elution peak volumes with low- to subfemtomole detection levels. In addition, a highly complex peptide mixture (outer membrane preparation from Psuedemonas aeruginosa) was efficiently separated with more than 100 proteins identified from a single reversed-phase LC-MS/MS analysis. Finally, the vented column (V-column) was utilized for on-line separations in a multidimensional chromatography/tandem MS experiment where large numbers of strong cation exchange chromatography fractions from a trypsinized yeast lysate were desalted, concentrated, and analyzed in a completely automated fashion. The procedures for constructing and using a V-column require minimal changes in current methods and equipment for nano-LC-MS analyses using columns of 100-microm diameter and smaller.     

Quantitative method for the analysis of tobacco-specific nitrosamines in cigarette tobacco and mainstream cigarette smoke by use of isotope dilution liquid chromatography tandem mass spectrometry

An improved liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for the determination of tobacco specific nitrosamines (TSNA). It utilizes four stable isotope-labeled internal standards instead of two as reported by others. A separate internal standard for each analyte is required to minimize sample matrix effects on each analyte, which can lead to poor analyte recoveries and decreases in method accuracy and precision if only one or two of the internal standards are used, especially for complex sample matrixes and when no sample cleanup steps are performed as in this study. In addition, two ion-transition pairs (instead of one) are used for each analyte for the confirmation and quantification, further enhancing the method's accuracy and robustness. These improvements have led to a new LC-MS/MS method that is faster, more sensitive, and selective than the traditional methods and more accurate and robust than the published LC-MS/MS methods. The linear range of the method was from 0.2 to 250 ng/mL with a limit of detection of each TSNA varied from 0.027 to 0.049 ng/mL. Good correlations between the results obtained by the new method and the traditional method were observed for the samples studied.     

Optimized time alignment algorithm for LC-MS data: correlation optimized warping using component detection algorithm-selected mass chromatograms

Correlation optimized warping (COW) based on the total ion current (TIC) is a widely used time alignment algorithm (COW-TIC). This approach works successfully on chromatograms containing few compounds and having a well-defined TIC. In this paper, we have combined COW with a component detection algorithm (CODA) to align LC-MS chromatograms containing thousands of biological compounds with overlapping chromatographic peaks, a situation where COW-TIC often fails. CODA is a variable selection procedure that selects mass chromatograms with low noise and low background (so-called "high-quality" mass chromatograms). High-quality mass chromatograms selected in each COW segment ensure that the same compounds (based on their mass and their retention time) are used in the two-dimensional benefit function of COW to obtain correct and optimal alignments (COW-CODA). The performance of the COW-CODA algorithm was evaluated on three types of complex data sets obtained from the LC-MS analysis of samples commonly used for biomarker discovery and compared to COW-TIC using a new global comparison method based on overlapping peak area: trypsin-digested serum obtained from cervical cancer patients, trypsin-digested serum from a single patient that was treated with varying preanalytical parameters (factorial design study), and urine from pregnant and nonpregnant women. While COW-CODA did result in minor misalignments in rare cases, it was clearly superior to the COW-TIC algorithm, especially when applied to highly variable chromatograms (factorial design, urine). The presented algorithm thus enables automatic time alignment and accurate peak matching of multiple LC-MS data sets obtained from complex body fluids that are often used for biomarker discovery.     

Reduction in matrix-related signal suppression effects in electrospray ionization mass spectrometry using on-line two-dimensional liquid chromatography.

The effect of liquid chromatographic separation on matrix-related signal suppression in electrospray ionization mass spectrometry (LC-ESI-MS) was investigated. A method incorporating on-line two-dimensional liquid chromatography mass spectrometry (LC/LC-MS) was developed to compensate for matrix effects and signal suppression in qualitative and quantitative analysis. The LC/LC-MS(MS) approach was successfully applied for single-component and multicomponent analysis in a variety of complex matrixes. It was demonstrated that matrix-related signal suppression could be induced solely by (i) column overload, (ii) matrix component-analyte coelution, or a combination of each. Application of on-line orthogonal LC/LC separations can be effective in reducing both causes of matrix-related signal suppression effects i.e., column overload and matrix-analyte coelution for a variety of LCn/MSn applications.     

ChromAlign: A two-step algorithmic procedure for time alignment of three-dimensional LC-MS chromatographic surfaces

We present an algorithmic approach to align three-dimensional chromatographic surfaces of LC-MS data of complex mixture samples. The approach consists of two steps. In the first step, we prealign chromatographic profiles: two-dimensional projections of chromatographic surfaces. This is accomplished by correlation analysis using fast Fourier transforms. In this step, a temporal offset that maximizes the overlap and dot product between two chromatographic profiles is determined. In the second step, the algorithm generates correlation matrix elements between full mass scans of the reference and sample chromatographic surfaces. The temporal offset from the first step indicates a range of the mass scans that are possibly correlated, then the correlation matrix is calculated only for these mass scans. The correlation matrix carries information on highly correlated scans, but it does not itself determine the scan or time alignment. Alignment is determined as a path in the correlation matrix that maximizes the sum of the correlation matrix elements. The computational complexity of the optimal path generation problem is reduced by the use of dynamic programming. The program produces time-aligned surfaces. The use of the temporal offset from the first step in the second step reduces the computation time for generating the correlation matrix and speeds up the process. The algorithm has been implemented in a program, ChromAlign, developed in C++ language for the .NET2 environment in WINDOWS XP. In this work, we demonstrate the applications of ChromAlign to alignment of LC-MS surfaces of several datasets: a mixture of known proteins, samples from digests of surface proteins of T-cells, and samples prepared from digests of cerebrospinal fluid. ChromAlign accurately aligns the LC-MS surfaces we studied. In these examples, we discuss various aspects of the alignment by ChromAlign, such as constant time axis shifts and warping of chromatographic surfaces.     

Sensitive detection of Bacillus anthracis spores by immunocapture and liquid chromatography-tandem mass spectrometry

Bacillus anthracis is one of the most dangerous agents of the bioterrorism threat. We present here a sensitive immuno-liquid chromatography-tandem mass spectrometry (immuno-LC-MS/MS) approach to spore detection in complex environmental samples. It is based on the combined specificity and sensitivity of two techniques: immunocapture and targeted mass spectrometry. The immunocapture step, realized directly on the intact spores, is essential for their selective isolation and concentration from complex environmental samples. After parallel trypsin and Glu-C digestions, proteotypic peptides corresponding to small acid-soluble spore protein-B (SASP-B) are specifically monitored in the multiple reaction monitoring (MRM) mass spectrometry mode. Peptide ratio is carefully monitored and provides an additional level of specificity, which is shown to be highly useful for distinguishing closely related samples and avoiding false-positive/negative results. Sensitivity at the level of the infectious dose is demonstrated, with limits of detection of 7 × 10(3) spores/mL of milk or 10 mg of soil. This mass spectrometry approach is thus complementary to polymerase chain reaction (PCR) techniques.     

Top-down identification and quantification of stable isotope labeled proteins from Aspergillus flavus using online nano-flow reversed-phase liquid chromatography coupled to a LTQ-FTICR mass spectrometer

Online liquid chromatography-mass spectrometric (LC-MS) analysis of intact proteins (i.e., top-down proteomics) is a growing area of research in the mass spectrometry community. A major advantage of top-down MS characterization of proteins is that the information of the intact protein is retained over the vastly more common bottom-up approach that uses protease-generated peptides to search genomic databases for protein identification. Concurrent to the emergence of top-down MS characterization of proteins has been the development and implementation of the stable isotope labeling of amino acids in cell culture (SILAC) method for relative quantification of proteins by LC-MS. Herein we describe the qualitative and quantitative top-down characterization of proteins derived from SILAC-labeled Aspergillus flavus using nanoflow reversed-phase liquid chromatography directly coupled to a linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (nLC-LTQ-FTICR-MS). A. flavus is a toxic filamentous fungus that significantly impacts the agricultural economy and human health. SILAC labeling improved the confidence of protein identification, and we observed 1318 unique protein masses corresponding to 659 SILAC pairs, of which 22 were confidently identified. However, we have observed some limiting issues with regard to protein quantification using top-down MS/MS analyses of SILAC-labeled proteins. The role of SILAC labeling in the presence of competing endogenously produced amino acid residues and its impact on quantification of intact species are discussed in detail.     

Analysis of histidine phosphorylation using tandem MS and ion-electron reactions

Phosphorylation of proteins is essential in intracellular signal transduction pathways in eukaryotic and prokaryotic cells. Histidine phosphorylation plays an important role in two-component signal transduction in bacteria. In this study, we describe the characterization of a synthetic histidine-phosphorylated peptide with four different mass spectrometric (MS) fragmentation techniques: Collision-induced dissociation (CID), electron capture dissociation, electron-transfer dissociation, and electron detachment dissociation. Furthermore, LC-MS methods were developed to detect histidine-phosphorylated peptides, which are acid-labile, in more complex samples. From these results, we concluded that nonacidic solvent systems or fast LC methods provide the best conditions for separation of histidine-phosphorylated peptides prior to electrospray ionization mass spectrometry analysis. Electron-based fragmentation methods should be used for determination of histidine phosphorylation sites, since CID results in very facile phosphate-related neutral losses. The developed LC-MS/MS methods were successfully applied to a tryptic digest of the cytoplasmic part of the histidine kinase EnvZ, which was in vitro autophosphorylated. Finally, a new method is described for nonretentive solid-phase extraction of histidine-phosphorylated peptides using polymeric Strata-X microcolumns.     

Clinical pharmacokinetic assessment of an anti-MAdCAM monoclonal antibody therapeutic by LC-MS/MS

Liquid chromatography tandem mass spectrometry (LC-MS/MS) has been shown to be a viable tool for preclinical pharmacokinetic (PK) analysis of monoclonal antibody (mAb) therapeutics. This work describes free and total PK assays for the mAb PF-00547,659 in serum of ulcerative colitis patients in a First-In-Human study [Vermeire, S. et al. Gut2011, 60 (8), 1068-1075]. The assay to measure free PF-00547,659 used immuno-enrichment with a biotinylated anti-idiotypic antibody and streptavidin magnetic beads. The total assay used enrichment by protein G magnetic beads. Following elution of PF-00547,659 from the beads, addition of an extended sequence stable isotope labeled peptide and trypsin digestion, a proteotypic peptide derived from the CDR region of the light chain of PF-00547,659 was quantified by LC-MS/MS. The free assay had a calibration range from 7.03 ng/mL to 450 ng/mL. The assay was precise and accurate with interbatch imprecision <16.5%, and interbatch inaccuracy <13.7% at all concentrations investigated during assay qualification. Results from LC-MS/MS methodologies are compared with historical immunoassay data originally acquired during the course of the clinical study. PK parameter estimates were highly correlated between the two analytical approaches. This work provides precedence that immunoaffinity LC-MS/MS can effectively be used to measure the serum concentrations of mAb therapeutics in clinical studies.     

Protocol for an electrospray ionization tandem mass spectral product ion library: development and application for identification of 240 pesticides in foods

Modern determination techniques for pesticides must yield identification quickly with high confidence for timely enforcement of tolerances. A protocol for the collection of liquid chromatography (LC) electrospray ionization (ESI)-quadruple linear ion trap (Q-LIT) mass spectrometry (MS) library spectra was developed. Following the protocol, an enhanced product ion (EPI) library of 240 pesticides was developed by use of spectra collected from two laboratories. A LC-Q-LIT-MS workflow using scheduled multiple reaction monitoring (sMRM) survey scan, information-dependent acquisition (IDA) triggered collection of EPI spectra, and library search was developed and tested to identify the 240 target pesticides in one single LC-Q-LIT MS analysis. By use of LC retention time, one sMRM survey scan transition, and a library search, 75-87% of the 240 pesticides were identified in a single LC/MS analysis at fortified concentrations of 10 ng/g in 18 different foods. A conventional approach with LC-MS/MS using two MRM transitions produced the same identifications and comparable quantitative results with the same incurred foods as the LC-Q-LIT using EPI library search, finding 1.2-49 ng/g of either carbaryl, carbendazim, fenbuconazole, propiconazole, or pyridaben in peaches; carbendazim, imazalil, terbutryn, and thiabendazole in oranges; terbutryn in salmon; and azoxystrobin in ginseng. Incurred broccoli, cabbage, and kale were screened with the same EPI library using three LC-Q-LIT and a LC-quadruple time-of-flight (Q-TOF) instruments. The library search identified azoxystrobin, cyprodinil, fludioxinil, imidacloprid, metalaxyl, spinosyn A, D, and J, amd spirotetramat with each instrument. The approach has a broad application in LC-MS/MS type targeted screening in food analysis.     

Synthesis/degradation ratio mass spectrometry for measuring relative dynamic protein turnover

One of the major unanswered questions in quantitative proteomics is that of dynamic protein turnover in the cell. Here we present a new approach to quantitative proteomics that measures the relative dynamic turnover of proteins in cellular systems. In this approach, termed synthesis/degradation ratio mass spectrometry, stable isotope labeling is employed to calculate a relative synthesis/degradation ratio that reflects the relative rate at which 13C is incorporated into individual proteins in the cell. This synthesis/degradation ratio calculation is based on a Poisson distribution model that is designed to support high-throughput analysis. Protein separation and analysis is accomplished by utilizing one-dimensional SDS-PAGE gel electrophoresis followed by cutting the gel into a series of bands for in-gel digestion. The resulting peptide mixtures are analyzed via solid-phase MALDI LC-MS and LC-MS/MS using a tandem time-of-flight mass spectrometer. A portion of the soluble protein fraction from an E. coli K-12 strain was analyzed with synthesis/degradation ratios varying from approximately 0.1 to 4.4 for a variety of different proteins. Unlike other quantitative techniques, synthesis/degradation ratio mass spectrometry requires only a single cell culture to obtain useful biological information about the processes occurring inside a cell. This technique is highly amenable to shotgun proteomics-based approaches and thus should allow relative turnover measurements for whole proteomes in the future.     

Identification of proteins in complexes by solid-phase microextraction/multistep elution/capillary electrophoresis/tandem mass spectrometry.

A method to directly identify proteins in complex mixtures by solid-phase microextraction (micro-SPE)/multistep elution/capillary electrophoresis (CE)/tandem mass spectrometry (MS/MS) is described. A sheathless liquid-metal junction interface is used to interface CE and electrospray ionization MS/MS. A subfemtomole detection limit is achieved for protein identification through database searching using MS/MS data. The SPE serves as a semiseparation dimension using an organic-phase step-elution gradient in combination with the second separation dimension for increased resolving power of complex peptide mixtures. This approach improves the concentration detection limit for CE and allows more proteins in complex mixtures to be identified. A 75-protein complex from yeast ribosome is analyzed using this method and 80-90% of the proteins in the complex can be identified by searching the database using the MS/MS data from a complete analysis. This multidimensional CE/MS/MS methodology provides an alternative to multidimensional liquid chromatography/MS/MS for direct identification of small amounts of protein in mixtures.