Identification of bacterial proteins observed in MALDI TOF mass spectra from whole cells.

Characteristic ions in the MALDI TOF mass spectra from bacterial cells have been associated with four known proteins. The proteins, observed both from cells and in filtered cellular suspensions, were isolated by HPLC and identified on the basis of their mass spectra and their partial amino acid sequence, determined using the Edman method (10-15 residues). The acid resistance proteins HdeA and HdeB give rise to ions near m/z 9735 and 9060 in MALDI TOF mass spectra from cells and from extracts of both Escherichia coli 1090 and Shigella flexneri PHS-1059. However, the proteins associated with proteolytic cleavage by the peptidase Lep, rather than the precursor proteins, were observed, both using cells and from cellular extracts. A cold-shock protein, CspA, was associated with the ion near m/z 7643 from Pseudomonas aeruginosa. Similarly, a cold-acclimation protein, CapB, was identified as the source of the ion near m/z 7684 in P. putida. This last protein was homologous with a known CapB from P. fragi. While these experiments involved the detection of known or homologous proteins from typical bacteria, this same approach could also be applied to the detection of unique proteins or biomarker proteins associated with other bacteria of public health significance.     

Accurate mass measurements for peptide and protein mixtures by using matrix-assisted laser desorption/ionization Fourier transform mass spectrometry

A new analytical scheme based on a combination of scanning FTMS, multiple-ion filling, and potential ramping methods has been developed for accurate molecular mass measurement of peptide and protein mixtures using broadband MALDI-FTMS. The scanning FTMS method alleviates the problems of time-of-flight effect for FTMS with an external MALDI ion source and provides a systematic means of sampling ions of different mass-to-charge ratios. The multiple-ion filling method is an effective way of trapping and retaining ions from successive ion generation/accumulation events. The potential ramping method allows the use of high trapping potentials for effective trapping of ions of high kinetic energies and the use of low trapping potentials for high-resolution detection of the trapped ions. With this analytical scheme, high-resolution broadband MALDI mass spectra covering a wide mass range of 1000-5700 Da were obtained. For peptide mixtures of mass range 1000-3500 Da, calibration errors of low part-per-millions were demonstrated using a parabolic calibration equation f2 = ML1/m2 + ML2/m + ML3, where f is the measured cyclotron frequency and ML1, ML2, and ML3 are calibration constants.     

Atmospheric pressure MALDI-fourier transform mass spectrometry

The coupling of atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) with Fourier transform mass spectrometry (FTMS) is described, and its significance for the high-resolution analysis of complex peptide mixtures is demonstrated. High kinetic energy and extensive metastable decay characteristic of ions generated by vacuum MALDI have been known to constitute a possible obstacle to high-resolution analysis by FTMS. Since the initial coupling of laser desorption techniques with FTMS was realized two decades ago, several different solutions have been proposed to control the energy of the ions and fulfill the promise of high sensitivity and high resolution offered by this analytical method. Initial results obtained on quadrupole time-of-flight and ion trap analyzers have shown that ions generated by MALDI at atmospheric pressure are intrinsically less energetic than those provided by vacuum MALDI. Our report indicates that this characteristic is particularly beneficial for FTMS applications in which a sharp reduction of metastable decay can make larger ion currents available for detection and possible tandem experiments. In our hands, AP MALDI-FTMS has enabled the analysis of complex peptide mixtures with resolution and accuracy comparable to those obtained by analogous electrospray ionization-FTMS experiments, with no evidence of either metastable decomposition or significant formation of matrix adducts. Analysis of a trypsin digest of bovine serum albumin provided signal-to-noise ratios and limits of detection similar to those obtained by ion trap analyzers, but with unmatched resolution and accuracy. AP MALDI has been shown to provide stable precursor ions in amounts that allowed for informative tandem experiments. Finally, the potential of AP MALDI-FTMS for the high-resolution screening of complex mixtures was demonstrated by the analysis of isobaric peptides differing in mass by less than 0.04 Da.     

Accurate mass-driven analysis for the characterization of protein phosphorylation. Study of the human Chk2 protein kinase

We describe the data-dependent analysis of protein phosphorylation using rapid-acquisition nano-LC-linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometry (nano-LC-FTMS). The accurate m/z values of singly, doubly, and triply charged species calculated from the theoretical protonated masses of peptides phosphorylated at all Ser, Thr, or Tyr residues of the human checkpoint 2 (Chk2) protein kinase were used for selected ion extraction and chromatographic analysis. Using a kinase-inactive Chk2 mutant as a control, accurate mass measurements from FTMS and collision-induced dissociation spectra, 11 novel Chk2 autophosphorylation sites were assigned. Additionally, the presence of additional Chk2 phosphorylation sites in two unique peptides was deduced from accurate mass measurements. Selected ion chromatograms of all Chk2 phosphopeptides gave single peaks except in three cases in which two closely eluting species were observed. These pairs of phosphopeptides were determined to be positional isomers from MS/MS analysis. In this study, it was also found that ions due to the neutral loss of phosphoric acid from the parent peptide ion were not prominent in 18 of 36 MS/MS spectra of O-linked Chk2 phosphopeptides. Thus, accurate mass-driven analysis and rapid parallel MS/MS acquisition is a useful method for the discovery of new phosphorylation sites that is independent of the signature losses from phosphorylated amino acid residues.     

MALDI ion trap mass spectrometer with charge detector for large biomolecule detection

Up to now, all commercial matrix-assisted laser desorption/ionization (MALDI) mass spectrometers still can not efficiently analyze very large biomolecules. In this work, we report the development of a novel MALDI ion trap mass spectrometer which can enrich biomolecular ions to enhance the detection sensitivity. A charge detector was installed to measure the large ions directly. With this design, we report the first measurement of IgM with the mass-to-charge ratio (m/z) at 980?000. In addition, quantitative measurements of the number of ions can be obtained. A step function frequency scan was first developed to get a clear signal in the m/z range from 200,000 to 1,000,000.     

Matrix-assisted laser desorption/ionization-mass spectrometry of hydrophobic proteins in mixtures using formic acid, perfluorooctanoic acid, and sorbitol

Three MALDI-MS sample/matrix preparation approaches were evaluated for their ability to enhance hydrophobic protein detection from complex mixtures: (1) formic acid-based formulations, (2) perfluorooctanoic acid (PFOA) surfactant addition, and (3) sorbitol addition. While MALDI-MS of Escherichia coli cells desorbed from a standard sinapinic acid matrix displayed 94 (M + H)+ ions, 119 were observed from a formic acid-based matrix with no more than 10 common to both. Formic acid matrix revealed many lipoproteins and an 8282 m/z ion proposed to be the abundant, water-insoluble ATPase proteolipid. Among the formic acid-based cocktails examined, the slowest rate of serine/threonine formylation was found for 50% H2O/33% 2-propanol/17% formic acid. Faster formylation was observed from cocktails containing more formic acid and from mixtures including CH3CN. Sinapinic, ferulic, DHB, 4-hydroxybenzylidene malononitrile, and 2-mercaptobenzothiazole matrixes performed well in formic acid formulations. Dramatic differences in mixture spectra were also observed from PFOA/sinapinic acid, at detergent concentrations exceeding the critical micelle concentration, although these matrix cocktails proved difficult to crystallize. E. coli ions observed from these matrix conditions are listed in Tables S-1 and S-3 (Supporting Information). Similar complementarity was observed for M. acetivorans whole-cell mixtures. Including sorbitol in the sinapinic acid matrix was found to promote homogeneous crystallization and to enhance medium and higher m/z ion detection from dilute E. coli cellular mixtures.     

Single cell matrix-assisted laser desorption/ionization mass spectrometry imaging

Application of mass spectrometry imaging (MS imaging) analysis to single cells was so far restricted either by spatial resolution in the case of matrix-assisted laser desorption/ionization (MALDI) or by mass resolution/mass range in the case of secondary ion mass spectrometry (SIMS). In this study we demonstrate for the first time the combination of high spatial resolution (7 μm pixel), high mass accuracy (<3 ppm rms), and high mass resolution (R = 100?000 at m/z = 200) in the same MS imaging measurement of single cells. HeLa cells were grown directly on indium tin oxide (ITO) coated glass slides. A dedicated sample preparation protocol was developed including fixation with glutaraldehyde and matrix coating with a pneumatic spraying device. Mass spectrometry imaging measurements with 7 μm pixel size were performed with a high resolution atmospheric-pressure matrix-assisted laser desorption/ionization (AP-MALDI) imaging source attached to an Exactive Orbitrap mass spectrometer. Selected ion images were generated with a bin width of Δm/z = ±0.005. Selected ion images and optical fluorescence images of HeLa cells showed excellent correlation. Examples demonstrate that a lower mass resolution and a lower spatial resolution would result in a significant loss of information. High mass accuracy measurements of better than 3 ppm (root-mean-square) under imaging conditions provide confident identification of imaged compounds. Numerous compounds including small metabolites such as adenine, guanine, and cholesterol as well as different lipid classes such as phosphatidylcholine, sphingomyelin, diglycerides, and triglycerides were detected and identified based on a mass spectrum acquired from an individual spot of 7 μm in diameter. These measurements provide molecularly specific images of larger metabolites (phospholipids) in native single cells. The developed method can be used for a wide range of detailed investigations of metabolic changes in single cells.     

Characterizing the phospholipid profiles in mammalian tissues by MALDI FTMS

Discussed here is an analytical method for profiling lipids and phospholipids directly from mammalian tissues excised from Mus musculus (house mouse). Biochemical analysis was accomplished through the use of matrix-assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry, where whole tissue sections of mouse brain, heart, and liver were investigated. Lipid and phospholipid ions create complex MALDI mass spectra containing multiple ions with different m/z values corresponding to the same fundamental chemical species. When a computational sorting approach is used to group these ions, the standard deviation for observed relative chemical abundance can be reduced to 6.02%. Relative standard deviations of 10% are commonly accepted for standard chromatographic phospholipid analyses. Average mass measurement accuracy for 232 spectra representing three tissue types from 12 specimens was calculated to be 0.0053 Da. Further it is observed, that the data and the analysis between all the animals have near-identical phospholipid contents in their brain, heart, and liver tissues, respectively. In addition to the need to accurately measure relative abundances of phospholipid species, it is essential to have adequate mass resolution for complete and accurate overall analysis. It is reasonable to make mass composition assignments with spectral resolving power greater than 8000. However, results from the present study reveal 14 instances (C12 carbon isotope) of multiple m/z ions having the same nominal value that require greater resolution in order that overlap will not occur. Spectra measured here have an average resolving power of 12 000. It is established that high mass resolution and mass accuracy coupled with MALDI ionization provide for rapid and accurate phospholipid analysis of mammalian tissue sections.     

Investigation of the unusual behavior of metolachlor under chemical ionization in a hybrid 3D ion trap mass spectrometer

This article describes the strange behavior of the widely used herbicide metolachlor under chemical ionization conditions in a hybrid source ion trap mass spectrometer in gas chromatography/mass spectrometry (GC/MS) coupling. With the use of ammonia as the reagent gas, metolachlor provides a chlorinated ion at m/z 295/297, almost as abundant as the protonated molecule at m/z 284/286, which cannot be isolated to perform tandem mass spectrometry (MS(n)) experiments. Curiously, this ion at m/z = M + 12 is not observed for the herbicides acetochlor and alachlor, which present very similar chemical structures. The chemical structure of the m/z 295/297 ions and the explanation of the observed phenomenon based on the metastable behavior of these ions were elucidated on the basis of experiments including isotopic labeling and modifications of the operating conditions of the ion trap mass spectrometer. This work allows one to give new recommendations for an optimized use of hybrid source ion trap mass spectrometers.     

Direct analysis and identification of Helicobacter and Campylobacter species by MALDI-TOF mass spectrometry.

Campylobacter jejuni, Campylobacter fetus, and Campylobacter coli were compared with Helicobacter pylori and Helicobacter mustelae by direct analysis of individual cultured colonies in 50% methanol-water with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS). H. pylori and Campylobacter species from blood agar culture produced unique, complex spectra with over 25 different ions in mass/charge (m/z) range from 2,000 to 62,000. A biomarker for H. pylori was centered around m/z 58,268, and H. mustelae was distinguished from H. pylori by its ions at m/z 49,608 and 57,231. Campylobacters could be distinguished from Helicobacters by their lack of ions around m/z 58,000 and 61,000 as well as distinguishing biomarkers of lower m/z: 10,074 and 25,478 for C. coli; m/z 10,285 and 12,901 for C. jejuni; m/z 10,726 and 11,289 for C. fetus. MALDI-TOF MS is a rapid and direct method for detection of these potentially pathogenic bacteria from culture.     

Discrimination between bacterial spore types using time-of-flight mass spectrometry and matrix-free infrared laser desorption and ionization

We demonstrate that molecular ions with mass-to-charge ratios (m/z) ranging from a few hundred to 19 050 can be desorbed from whole bacterial spores using infrared laser desorption and no chemical matrix. We have measured the mass of these ions using time-of-flight mass spectrometry and we observe that different ions are desorbed from spores of Bacillus cereus, Bacillus thuringiensis, Bacillus subtilis, and Bacillus niger. Our results raise the possibility of identifying microorganisms using mass spectrometry without conventional sample preparation techniques such as the addition of a matrix. We have measured the dependence of the ion yield from B. subtilis on desorption wavelength over the range 3.05-3.8 microm, and we observe the best results at 3.05 microm. We have also generated mass spectra from whole spores using 337-nm ultraviolet laser desorption, and we find that these spectra are inferior to spectra generated with infrared desorption. Since aerosol analysis is a natural application for matrix-free desorption, we have measured mass spectra from materials such as ragweed pollen and road dust that are likely to form a background to microbial aerosols. We find that these materials are readily differentiated from bacterial spores.     

Scrubbing ions with molecules: kinetic studies of chemical noise reduction in mass spectrometry using ion-molecule reactions with dimethyl disulfide

The kinetics and product distributions of the reactions of dimethyl disulfide (DMDS) have been investigated with a group of chemical background ions commonly observed in atmospheric pressure ionization (API) mass spectrometry (MS) in order to assess the value of this molecule in filtering (or "scrubbing") these ions by changing their mass/charge (m/z) ratio. The measurements were taken with a novel electrospray ionization/selected ion flow tube/QqQ tandem mass spectrometer. The background ions studied include those with m/z 42 (protonated acetonitrile, ACN), 83 (protonated ACN dimer), 99 (protonated phosphoric acid), 117 (water cluster of m/z 99), 131 (methanol cluster of m/z 99), 149 (protonated phthalic anhydride, formed from the phthalates), and 327 (protonated triphenyl phosphate). In addition, reactions of DMDS have been studied with two model analytes--protonated caffeine and doubly protonated bradykinin--in order to assess the selectivity of DMDS reactivity. All the measurements were taken at 295 +/- 2 K in helium buffer gas at a pressure of 0.35 +/- 0.01 Torr. DMDS was observed to react efficiently with m/z 42 (ACNH+), 149 (from phthalates), and 99 (protonated phosphoric acid), with k/kc=0.91, 0.47, and 0.38, respectively. Only proton transfer was observed with ACNH+, followed by the secondary reaction of [DMDSH]+ with DMDS to yield [CH3S-S(CH3)-SCH3]+. Ligation of DMDS was the dominant primary channel observed for the reaction of the m/z 149 background ion; however, some proton transfer also was observed. Both of these primary product ions react further with DMDS to yield [CH3S-S(CH3)-SCH3]+, the structure of which we have determined computationally using DFT calculations. Only the sequential ligation with two DMDS molecules was observed for the reaction of the m/z 99 ion. Reactions of DMDS with m/z 117 [H3PO4 + H + H2O]+ and m/z 131 [H3PO4 + H + MeOH]+ were observed to proceed with k/kc=0.71 and 0.058, respectively. Ligand substitution of DMDS for H2O predominated ( approximately 94%) over DMDS ligation ( approximately 6%) in the reaction with m/z 117, while only DMDS ligation was observed for the reaction of m/z 131 with DMDS. In contrast, the reactions of DMDS with ions of m/z 83 (protonated dimer of ACN) and 327 (protonated triphenyl phosphate) were extremely inefficient, with k/kc=0.0042 and 0.0079, respectively. The higher reactivity of DMDS toward ACNH+ (m/z 42) compared to (ACN)2H+ (m/z 83) is attributed to the lower proton affinity of the unsolvated ACN. The reactivity of DMDS toward the two model analyte ions studied-protonated caffeine and doubly protonated bradykinin-was negligible, with k/kc=0.0073 and 0.010, for the respective reactions. These results suggest that, under appropriate reagent pressure conditions, DMDS can be an appropriate reagent for chemically filtering out many common API-MS background ions, without significantly affecting the observed intensity of analyte peaks.     

Identification of microcystin toxins from a strain of Microcystis aeruginosa by liquid chromatography introduction into a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer

The cyclic heptapeptide microcystin toxins produced by a strain of Microcystis aeruginosa that has not been investigated previously were separated by liquid chromatography and identified by high-accuracy m/z measurements of their [M + H]+ ions and the fragment ions produced by collision-activated dissociation of the [M + H]+ ions. The cyanobacteria B2666 strain was cultured in a standard growth medium, and the toxins were released from the cells, extracted from the aqueous phase, and concentrated using standard procedures. The microcystins were separated by reversed-phase microbore liquid chromatography and introduced directly into a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer with electrospray ionization. The known microcystins (MC) MC-LR, MC-LA, [MeSer7]MC-LR, MC-LL, MC-LF, and MC-L(Aba) were identified along with the two previously unreported structural variants [Asp3]MC-LA and [Asp3]MC-LL. In addition to the [M + H]+ ions, accurate m/z measurements were made of 12-18 product ions for each identified microcystin. The mean difference between measured and calculated exact m/z was less than 2 parts per million, which often allowed assignment of unique compositions to the observed ions. A mechanism is presented that accounts for an important collision-activated dissociation process that gives valuable sequence ions from microcystins that do not contain arginine. The analytical technique used in this work is capable of supporting fairly rapid and very reliable identifications of known microcystins when standards are not available and of most structural variants independent of additional information from other analytical techniques.     

Rapid profiling of induced proteins in bacteria using MALDI-TOF mass spectrometric detection of nonporous RP HPLC-separated whole cell lysates.

A method for rapid profiling of water-soluble proteins from whole cell lysates has been developed using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) following separation by reversed-phase high-performance liquid chromatography (RP HPLC). Rapid separation of proteins from cell lysates was achieved using columns packed with C18 nonporous (NP) silica beads. Using this method, the whole cell lysate water-soluble proteins of E. coli were separated in under 15 min. A method using two columns in series at different temperatures was used in order to provide high loadability without loss of separation efficiency. The nonporous packing in the columns provided for high recovery. Eluting fractions were collected and analyzed by MALDI-TOFMS to determine the molecular weights and peptide maps of the proteins. These methods provided for the rapid screening and identification of proteins from E. coli where the response of E. coli to L-arabinose induction was studied. In this work, it is demonstrated that NP RP HPLC with MALDI-TOFMS detection may serve as a rapid means of detecting and identifying changes in bacterial protein expression due to external stimuli.     

Differentiation of isomeric conjugated bile acids using positive-ion B/E linked scans.

The main objective of this work was to use positive-ion fast atom bombardment mass spectrometry (FAB-MS) B/E linked scan spectra to investigate the possibility of differentiating positional isomers of various authentic glycine- and taurine-conjugated bile acids. Sodium salts of 14 conjugated bile acids were individually ionized by FAB-MS and characterized by scanning simultaneously the magnetic field B and the electric sector field E such that B/E remained constant throughout the scan. The dominant fragment ions could be related to cleavage of the aliphatic side chain with charge retention on the conjugated end of the bile acids. However, fragment ions arising from ring cleavages were also observed and could be used to distinguish the positions of substituent hydroxyl groups. For example, ring cleavage of conjugated dihydroxy bile acids at C-7/C-8 and C-9/C-10 permitted the differentiation of chenodeoxycholyltaurine (3 alpha,7 alpha-substitution pattern) from deoxycholyltaurine (3 alpha,12 alpha-substitution pattern) based on the presence of fragment ions at m/z 388 or m/z 404, which were indicative of hydroxyl group substitutions at either the 7- or 12-positions, respectively. It was concluded that B/E linked scans can be used to discriminate positional isomers of conjugated bile acids.     

Primary structure determination of peptides and enzymatically digested proteins using capillary liquid chromatography/mass spectrometry and rapid linked-scan techniques

Primary protein sequences were determined for both peptides and enzymatically digested proteins by rapid linked-scan (B/E) liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) at the low-picomole level (10-50 pmol). During the course of a single LC/MS/MS analysis, we demonstrated that it is possible to generate interpretable collision-induced dissociation spectra of the eluting proteolytic peptides. Molecular weights of tryptic peptides were established by using 1/10 of the protein digest by operating in the capillary LC/frit-FABMS mode. Peptides exhibiting the strongest MH+ ions were then selected for subsequent LC/MS/MS analysis (typically 1/5 of the remaining protein digest). Elution times for each chromatographic peak were generally greater than 30 s. It was therefore possible to obtain a minimum of six B/E fast linked-scan spectra during the course of elution of each peptide component. Typically, B/E linked scans of the greatest ion abundance (obtained at the chromatographic peak maximum) were averaged to enhance the signal/noise ratio at these low-picomole levels. Unit resolution was observed for product ions below m/z 1000. Rapid linked scanning by LC/frit-FABMS/MS provided mass assignments for product ions within 0.2-0.3 amu of theoretical values. Side-chain fragment ions (wn and dn) were also observed, which allowed for the differentiation of isobaric amino acids (e.g., leucine and isoleucine). Examples of the application of this fast linked-scan technique to LC/MS/MS are presented for complex mixtures of unknown peptides and the tryptic digestion of phosphorylated beta-casein.     

Measuring the mass of an electron by LC/TOF-MS: a study of "twin ions"

While investigating the in-source CID fragmentation of nonsteroidal antiinflammatory drugs (NSAIDs), it was noticed that the same fragment ion (nominal mass) formed in either positive or negative ion electrospray for a suite of NSAIDs. For example, ibuprofen with a molecular mass of 206, fragments to the m/z 161 ion in negative ion from its deprotonated molecule (m/z 205, [M - H]-) and fragments to the m/z 161 ion in positive ion from its protonated molecule (m/z 207, [M + H]+). This fragment ion was euphemistically called a "twin ion"because of the same nominal mass despite opposite charge. The CID fragmentation of the twin ions was confirmed also by LC/MS/MS ion trap. Accurate mass measurements in negative ion show that the loss was due to CO2 (measured loss of 43.9897 atomic mass units (u) versus calculated loss of 43.9898 u for N = 10) and in positive ion the loss is due to HCOOH (measured loss of 46.0048 u versus calculated loss of 46.0055 u, N = 10). It was realized that, in fact, the ions were not "identical mass twins of opposite charge" but separated in accurate mass by two electrons. The accurate mass measurement by liquid chromatography/time-of-flight-mass spectrometry (LC/TOF-MS) can distinguish between the two fragment ions of ibuprofen (161.13362 +/- 0.00019 and 161.13243 +/- 0.00014 for N = 20). This experiment was repeated for two other NSAIDs, and the mass of an electron was measured as the difference between the twin ions, which was 0.00062 u +/- 14.8% relative standard deviation (N = 20 analyses). Thus, the use of continuous calibration makes it possible to measure the mass of an electron within one significant figure using the NSAID solution. This result shows the importance of including electron mass in accurate mass measurements and the value of a benchmark test for LC/TOF-MS mass accuracy.     

Study of the fragmentation mechanism of protonated 6-hydroxychlorzoxazone: application in simultaneous analysis of CYP2E1 activity with major human cytochrome P450s

The application of liquid chromatography tandem mass spectrometry for simultaneous analysis of major human cytochrome P450 activities via a single atmospheric pressure ionization (API) LC/MS/MS method has been hampered by the preferred detection of 6-hydroxychlorzoxazone (HCZ), the metabolite of the CYP2E1 probe, chlorzoxazone, under negative API. An initial simulation of the dissociation constants suggested the potential ionization of the enol form of HCZ at low pH, and the accurate mass measurements confirmed the presence of the protonated HCZ signal under (+) ESI at pH 3. However, the CID spectrum of the protonated HCZ resulted in a few intense, but uncommon, fragment ions that could be utilized for specific selected reaction monitoring (SRM) transitions. The deduced elemental compositions of these fragment ions indicated possible aromatic ring opening for the first two intense product ions at m/z 130 and 115, as well as chlorine radical loss for the third ion at m/z 151. Further precursor and product ion scan studies, along with the deuterium ion exchange in solution, revealed the involvement of three distinct pathways of fragmentation. The m/z 186-->130 transition, which was shown to be specific in human plasma and rat hepatic microsomes, was further combined with the SRM transition of reserpine (internal standard) and eight probe substrates for human cytochrome P450 isoforms. This led to the development of a full LC/MS/MS method capable of analyzing a total of nine human P450 activities within 3 min, including CYP2E1, using a single assay in the (+) ESI mode. The HCZ assay showed excellent linearity with a coefficient of determination (R2) greater than 0.98 at dynamic range of 0.05 (LOQ) to 40 microM. Preliminary data from the three-day validation of the HCZ assay indicated that the accuracy and precision for quality control samples was within +/- 15% of the spiked concentration at all levels.     

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.