Improved precision of iTRAQ and TMT quantification by an axial extraction field in an Orbitrap HCD cell

Improving analytical precision is a major goal in quantitative differential proteomics as high precision ensures low numbers of outliers, a source of false positives with regard to quantification. In addition, higher precision increases statistical power, i.e., the probability to detect significant differences. With chemical labeling using isobaric tags for relative and absolute quantitation (iTRAQ) or tandem mass tag (TMT) reagents, quantification is based on the extraction of reporter ions from tandem mass spectrometry (MS/MS) spectra. We compared the performance of two versions of the LTQ Orbitrap higher energy collisional dissociation (HCD) cell with and without an axial electric field with regard to reporter ion quantification. The HCD cell with the axial electric field was designed to push fragment ions into the C-trap and this version is mounted in current Orbitrap XL ETD and Orbitrap Velos instruments. Our goal was to evaluate whether the purported improvement in ion transmission had a measurable impact on the precision of MS/MS based quantification using peptide labeling with isobaric tags. We show that the axial electric field led to an increased percentage of HCD spectra in which the complete set of reporter ions was detected and, even more important, to a reduction in overall variance, i.e., improved analytical precision of the acquired data. Notably, adequate precision of HCD-based quantification was maintained even for low precursor ion intensities of a complex biological sample. These findings may help researchers in their design of quantitative proteomics studies using isobaric tags and establish HCD-based quantification on the LTQ Orbitrap as a highly precise approach in quantitative proteomics.     

Comparative evaluation of two isobaric labeling tags, DiART and iTRAQ

Isobaric tags have broad applications in both basic and translational research, as demonstrated by the widely used isobaric tag for relative and absolute quantitation (iTRAQ). Recent results from large-scale quantitative proteomics projects, however, indicate that protein quantification by iTRAQ is often biased in complex biological samples. Here, we report the application of another isobaric tag, deuterium isobaric amine reactive tag (DiART), for quantifying the proteome of Thermoanaerobacter tengcongensis (T. tengcongensis), a thermophilic bacterium first discovered in China. We compared the performance of DiART with iTRAQ from several different aspects, including their fragmentation mechanisms, the number of identified proteins, and the accuracy of quantification. Our results revealed that, as compared with iTRAQ, DiART yielded significantly stronger reporter ions, which did not reduce the number of identifiable peptides, but improved the signal-to-noise ratio (S/N) for quantification. Remarkably, we found that, under identical chromatography and mass spectrometry (MS) conditions, DiART exhibited less reporter ions ratio compression than iTRAQ, probably due to more reporter ions with higher intensities produced by DiART labeling. Taken together, we demonstrate that DiART is a valuable alternative of iTRAQ with enhanced performance for quantitative proteomics.     

Stable-isotope dimethyl labeling for quantitative proteomics

In this paper, we report a novel, stable-isotope labeling strategy for quantitative proteomics that uses a simple reagent, formaldehyde, to globally label the N-terminus and epsilon-amino group of Lys through reductive amination. This labeling strategy produces peaks differing by 28 mass units for each derivatized site relative to its nonderivatized counterpart and 4 mass units for each derivatized isotopic pair. This labeling reaction is fast (less than 5 min) and complete without any detectable byproducts based on the analysis of MALDI and LC/ESI-MS/MS spectra of both derivatized and nonderivatized peptide standards and tryptic peptides of hemoglobin molecules. The intensity of the a(1) and y(n-1) ions produced, which were not detectable from most of the nonderivatized fragments, was substantially enhanced upon labeling. We further tested the method based on the analysis of an isotopic pair of peptide standards and a pair of defined protein mixtures with known H/D ratios. Using LC/MS for quantification and LC/MS/MS for peptide sequencing, the results show a negligible isotopic effect, a good mass resolution between the isotopic pair, and a good correlation between the experimental and theoretical data (errors 0-4%). The relative standard deviation of H/D values calculated from peptides deduced from the same protein are less than 13%. The applicability of the method for quantitative protein profiling was also explored by analyzing changes in nuclear protein abundance in an immortalized E7 cell with and without arsenic treatment.     

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.     

High-throughput comparative proteome analysis using a quantitative cysteinyl-peptide enrichment technology

A new quantitative cysteinyl-peptide enrichment technology (QCET) was developed to achieve higher efficiency, greater dynamic range, and higher throughput in quantitative proteomics that use stable-isotope labeling techniques combined with high-resolution liquid chromatography (LC)-mass spectrometry (MS). This approach involves (18)O labeling of tryptic peptides, high-efficiency enrichment of cysteine-containing peptides, and confident protein identification and quantification using the accurate mass and time tag strategy. Proteome profiling of na?ve and in vitro-differentiated human mammary epithelial cells using QCET resulted in the identification and quantification of 603 proteins in a single LC-Fourier transform ion cyclotron resonance MS analysis. Advantages of this technology include the following: (1) a simple, highly efficient method for enriching cysteinyl-peptides; (2) a high-throughput strategy suitable for extensive proteome analysis; and (3) improved labeling efficiency for better quantitative measurements. This technology enhances both the functional analysis of biological systems and the detection of potential clinical biomarkers.     

Tandem mass tag protein labeling for top-down identification and quantification

Top-down mass spectrometry holds tremendous potential for characterization and quantification of intact proteins. So far, however, very few studies have combined top-down proteomics with protein quantification. In view of the success of isobaric mass tags in quantitative bottom-up proteomics, we applied the tandem mass tag (TMT) technology to label intact proteins and examined the feasibility to directly quantify TMT-labeled proteins. A top-down platform encompassing separation via ion-pair reversed-phase liquid chromatography using monolithic stationary phases coupled online to an LTQ-Orbitrap Velos electron-transfer dissociation (ETD) mass spectrometer (MS) was established to simultaneously identify and quantify TMT-labeled proteins. The TMT-labeled proteins were found to be readily dissociated under high-energy collision dissociation (HCD) activation. The liberated reporter ions delivered expected ratios over a wide dynamic range independent of the protein charge state. Furthermore, protein sequence tags generated either by low-energy HCD or ETD activation along with the intact protein mass information allow for confident identification of small proteins below 35 kDa. We conclude that the approach presented in this pilot study paves the way for further developments and numerous applications for straightforward, accurate, and multiplexed quantitative analysis in protein chemistry and proteomics.     

Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags

A new 6-plex isobaric mass tagging technology is presented, and proof of principle studies are carried out using standard protein mixtures and human cerebrospinal fluid (CSF) samples. The Tandem Mass Tags (TMT) comprise a set of structurally identical tags which label peptides on free amino-terminus and epsilon-amino functions of lysine residues. During MS/MS fragmentation, quantification information is obtained through the losses of the reporter ions. After evaluation of the relative quantification with the 6-plex version of the TMT on a model protein mixture at various concentrations, the quantification of proteins in CSF samples was performed using shotgun methods. Human postmortem (PM) CSF was taken as a model of massive brain injury and comparison was carried out with antemortem (AM) CSF. After immunoaffinity depletion, triplicates of AM and PM CSF pooled samples were reduced, alkylated, digested by trypsin, and labeled, respectively, with the six isobaric variants of the TMT (with reporter ions from m/z = 126.1 to 131.1 Th). The samples were pooled and fractionated by SCX chromatography. After RP-LC separation, peptides were identified and quantified by MS/MS analysis with MALDI TOF/TOF and ESI-Q-TOF. The concentration of 78 identified proteins was shown to be clearly increased in PM CSF samples compared to AM. Some of these proteins, like GFAP, protein S100B, and PARK7, have been previously described as brain damage biomarkers, supporting the PM CSF as a valid model of brain insult. ELISA for these proteins confirmed their elevated concentration in PM CSF. This work demonstrates the validity and robustness of the tandem mass tag (TMT) approach for quantitative MS-based proteomics.     

Carbonyl-reactive tandem mass tags for the proteome-wide quantification of N-linked glycans

N-Linked protein glycosylation is one of the most prevalent post-translational modifications and is involved in essential cellular functions such as cell-cell interactions and cellular recognition as well as in chronic diseases. In this study, we explored stable isotope labeled carbonyl-reactive tandem mass tags (glyco-TMTs) as a novel approach for the quantification of N-linked glycans. Glyco-TMTs bearing hydrazide- and aminooxy-functionalized groups were compared for glycan reducing end derivatization efficiency and quantification merits. Aminooxy TMTs outperform the hydrazide reagents in terms of labeling efficiency (>95% vs 65% at 0.1 μM) and mass spectrometry based quantification using heavy/light-TMT labeled glycans enabled accurate quantification in MS1 spectra (CV < 15%) over a broad dynamic range (up to 1:40). In contrast, isobaric TMT labeling with quantification of reporter ions in tandem mass spectra suffered from severe ratio compression already at low sample ratios. To demonstrate the practical utility of the developed approach, we characterized the global N-linked glycosylation profiles of the isogenic human colon carcinoma cell lines SW480 (primary tumor) and SW620 (metastatic tumor). The data revealed significant down-regulation of high-mannose glycans in the metastatic cell line.     

Stable-isotope dimethylation labeling combined with LC-ESI MS for quantification of amine-containing metabolites in biological samples

One of the challenges associated with metabolome profiling in complex biological samples is to generate quantitative information on the metabolites of interest. In this work, a targeted metabolome analysis strategy is presented for the quantification of amine-containing metabolites. A dimethylation reaction is used to introduce a stable isotopic tag onto amine-containing metabolites followed by LC-ESI MS analysis. This labeling reaction employs a common reagent, formaldehyde, to label globally the amine groups through reductive amination. The performance of this strategy was investigated in the analysis of 20 amino acids and 15 amines by LC-ESI MS. It is shown that the labeling chemistry is simple, fast (<10-min reaction time), specific, and provides high yields under mild reaction conditions. The issue of isotopic effects of the labeled amines on reversed-phase (RP) and hydrophilic interaction (HILIC) LC separations was examined. It was found that deuterium labeling causes an isotope effect on the elution of labeled amines on RPLC but has no effect on HILIC LC. However, 13C-dimethylation does not show any isotope effect on either RPLC or HILIC LC, indicating that 13C-labeling is a preferred approach for relative quantification of amine-containing metabolites in different samples. The isotopically labeled 35 amine-containing analogues were found to be stable and proved to be effective in overcoming matrix effects in both relative and absolute quantification of these analytes present in a complicated sample, human urine. Finally, the characteristic mass difference provides additional structural information that reveals the existence of primary or secondary amine functional groups in amine-containing metabolites. As an example, for a human urine sample, a total of 438 pairs of different amine-containing metabolites were detected, at signal-to-noise ratios of greater than 10, by using the labeling strategy in conjunction with RP LC-ESI Fourier-transform ion cyclotron resonance MS.     

Application of metal-coded affinity tags (MeCAT): absolute protein quantification with top-down and bottom-up workflows by metal-coded tagging

As the quantification of peptides and proteins extends from comparative analyses to the determination of actual amounts, methodologies for absolute protein quantification are desirable. Metal-coded affinity tags (MeCAT) are chemical labels for peptides and proteins with a lanthanide-bearing chelator as a core. This modification of analytes with non-naturally occurring heteroelements adds the analytical possibilities of inductively coupled plasma mass spectrometry (ICPMS) to quantitative proteomics. We here present the absolute quantification of recombinantly expressed aprotinin out of its host cell protein background using two independent MeCAT methodologies. A bottom-up strategy employs labeling of primary amino groups on peptide level. Synthetic peptides with a MeCAT label which are externally quantified by flow injection analysis (FIA)-ICPMS serve as internal standard in nanoHPLC-ESI-MS/MS. In the top-down approach, protein is labeled on cysteine residues and separated by two-dimensional gel electrophoresis. Flow injection analysis of dissolved gel spots by ICPMS yields the individual protein amount via its lanthanide label content. The enzymatic determination of the fusion protein via its β-galactosidase activity found 8.3 and 9.8 ng/μg (nanogram fusion protein per microgram sample) for batches 1 and 2, respectively. Using MeCAT values of 4.0 and 5.4 ng/μg are obtained for top-down analysis, while 14.5 and 15.9 ng/μg were found in the bottom-up analysis.     

Investigation of sialylation aberration in N-linked glycopeptides by lectin and tandem labeling (LTL) quantitative proteomics

The accuracy in quantitative analysis of N-linked glycopeptides and glycosylation site mapping in cancer is critical to the fundamental question of whether the aberration is due to changes in the total concentration of glycoproteins or variations in the type of glycosylation of proteins. Toward this goal, we developed a lectin-directed tandem labeling (LTL) quantitative proteomics strategy in which we enriched sialylated glycopeptides by SNA, labeled them at the N-terminus by acetic anhydride ((1)H(6)/(2)D(6)) reagents, enzymatically deglycosylated the differentially labeled peptides in the presence of heavy water (H(2)(18)O), and performed LC/MS/MS analysis to identify glycopeptides. We successfully used fetuin as a model protein to test the feasibility of this LTL strategy not only to find true positive glycosylation sites but also to obtain accurate quantitative results on the glycosylation changes. Further, we implemented this method to investigate the sialylation changes in prostate cancer serum samples as compared to healthy controls. Herein, we report a total of 45 sialylated glycopeptides and an increase of sialylation in most of the glycoproteins identified in prostate cancer serum samples. Further quantitation of nonglycosylated peptides revealed that sialylation is increased in most of the glycoproteins, whereas the protein concentrations remain unchanged. Thus, LTL quantitative technique is potentially an useful method for obtaining simultaneous unambiguous identification and reliable quantification of N-linked glycopeptides.     

High-Resolution Enabled TMT 8-plexing

Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low-mass range of tandem MS spectra for relative quantification. The number of samples that can be compared in a single experiment (multiplexing) is limited by the number of different reporter ions that can be generated by differential stable isotope incorporation ((15)N, (13)C) across the reporter and the mass balancing parts of the reagents. Here, we demonstrate that a higher multiplexing rate can be achieved by utilizing the 6 mDa mass difference between (15)N- and (13)C-containing reporter fragments, in combination with high-resolution mass spectrometry. Two variants of the TMT127 and TMT129 reagents are available; these are distinguished by the position and the nature of the incorporated stable isotope in the reporter portions of the labels (TMT127L, (12)C(8)H(16)(15)N(1)(+); TMT127H, (12)C(7)(13)C(1)H(16)(14)N(1)(+); TMT129L, (12)C(6)(13)C(2)H(16)(15)N(1)(+); and TMT129H, (12)C(5)(13)C(3)H(16)(14)N(1)(+)). We demonstrate that these variants can be baseline-resolved in Orbitrap Elite higher-energy collision-induced dissociation spectra recorded with a 96 ms transient enabling comparable dynamic range, precision, and accuracy of quantification as 1 Da spaced reporter ions. The increased multiplexing rate enabled determination of inhibitor potencies in chemoproteomic kinase assays covering a wider range of compound concentrations in a single experiment, compared to conventional 6-plex TMT-based assays.     

Isobaric peptide termini labeling utilizing site-specific N-terminal succinylation

Recently, we introduced a novel approach for protein quantification based on isobaric peptide termini labeling (IPTL). In IPTL, both peptide termini are dervatized in two separate chemical reactions with complementary isotopically labeled reagents to generate isobaric peptide pairs. Here, we describe a novel procedure for the two chemical reactions to enable a cost-effective and rapid method. We established a selective N-terminal peptide modification reaction using succinic anhydride. Dimethylation was used as second chemical reaction to derivatize lysine residues. Both reactions can be performed within 15 min in one pot, and micropurification of the peptides between the two reactions was not necessary. For data analysis, we developed the force-find algorithm in IsobariQ which searches for corresponding peaks to build up peak pairs in tandem mass spectrometry (MS/MS) spectra where Mascot could not identify opposite sequences. Utilizing force-find, the number of quantified proteins was improved by more than 50% in comparison to the standard data analysis in IsobariQ. This was applied to compare the proteome of HeLa cells incubated with S-trityl-L-cysteine (STLC) to induce mitotic arrest and apoptosis. More than 50 proteins were found to be quantitatively changed, and most of them were previously reported in other proteome analyses of apoptotic cells. Furthermore, we showed that the two complementary isotopic labels coelute during liquid chromatography (LC) separation and that the linearity of relative IPTL quantification is not affected by a complex protein background. Combining the optimized reactions for IPTL with the open source data analysis software IsobariQ including force-find, we present a straightforward and rapid approach for quantitative proteomics.     

Fluorescein as a versatile tag for enhanced selectivity in analyzing cysteine-containing proteins/peptides using mass spectrometry

Fluorescence-based tagging in proteomics is useful in tracking and quantifying target proteins during sample preparation or chromatographic processes. In this study, we report a novel cysteinyl tagging method using a popular fluorophore, fluorescein derivative. Such visible dyes were shown to have multiple unique characteristics, including a unique reporter ion containing the dye moiety caused by collision-induced dissociation (CID) and high affinity toward multicarboxylate functional groups, which could be useful for enhanced selectivity in MS-based proteomics. We used sulfhydryl-reactive 5-iodoacetamidofluorescein to target cysteinyl residues on the intact protein of ovalbumin and bovine serum albumin as well as proteins in MCF-7 cells. After trypsin digestion, the digests were analyzed by nanoLC-ESI-Q-TOF or MALDI-TOF. The resulting MS spectra of tryptic fragments were similar to those of unlabeled or iodoacetamide-derivatized proteins, and the MS/MS fragmentation of all fluorescein-tagged peptides was readily interpretable with intact label. Thus, fluorescein-derivatized proteins can be identified by automatic mass mapping or peptide sequencing with high confidence. It is notable that, in MS/MS mode, a strong reporter ion (m/z 422) containing the fluorescein moiety was readily detected and was believed to derive from the immonium fragment of fluorescein-labeled cysteine residues, f C (m/z 463), under CID conditions. Using a precursor scan of the reporter ion, a cysteinyl protein, ovomucoid, was identified to be present in the ovalbumin sample as an impurity. The fluorescein derivatives were further shown to have high affinities toward metal-chelating materials that have iminodiacetic acid functional groups either with or without the presence of bound metal ions. When coupling with stable isotope dimethyl labeling, fluorescein-tagged peptides could be selectively enriched, identified, and quantified. In view of its popularity, visible tracking, and unique characteristics for developing selective methods, fluorescein tagging holds great promises for targeting proteomics.     

De novo sequencing of neuropeptides using reductive isotopic methylation and investigation of ESI QTOF MS/MS fragmentation pattern of neuropeptides with N-terminal dimethylation

A stable-isotope dimethyl labeling strategy was previously shown to be a useful tool for quantitative proteomics. More recently, N-terminal dimethyl labeling was also reported for peptide sequencing in combination with database searching. Here, we extend these previous studies by incorporating N-terminal isotopic dimethylation for de novo sequencing of neuropeptides directly from tissue extract without any genomic information. We demonstrated several new sequencing applications of this method in addition to the identification of the N-terminal residue using the enhanced a(1) ion. The isotopic labeling also provides easier and more confident de novo sequencing of peptides by comparing similar MS/MS fragmentation patterns of the isotopically labeled peptide pairs. The current study on neuropeptides shows several distinct fragmentation patterns after N-terminal dimethylation which have not been reported previously. The y((n-1)) ion is enhanced in multiply charged peptides and is weak or missing in singly charged peptides. The MS/MS spectra of singly charged peptides are simplified due to the enhanced N-terminal fragments and suppressed internal fragments. The neutral loss of dimethylamine is also observed. The mechanisms for the above fragmentations are proposed. Finally, the structures of the immonium ion and related ions of N(alpha), N(epsilon)-tetramethylated lysine and N(epsilon)-dimethylated lysine are explored.     

Stable isotope labeling by essential nutrients in cell culture for preparation of labeled coenzyme A and its thioesters

Stable isotope dilution mass spectrometry (MS) represents the gold standard for quantification of endogenously formed cellular metabolites. Although coenzyme A (CoA) and acyl-CoA thioester derivatives are central players in numerous metabolic pathways, the lack of a commercially available isotopically labeled CoA limits the development of rigorous MS-based methods. In this study, we adapted stable isotope labeling by amino acids in cell culture (SILAC) methodology to biosynthetically generate stable isotope labeled CoA and thioester analogues for use as internal standards in liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) assays. This was accomplished by incubating murine hepatocytes (Hepa 1c1c7) in media in which pantothenate (a precursor of CoA) was replaced with [(13)C(3)(15)N(1)]-pantothenate. Efficient incorporation into various CoA species was optimized to >99% [(13)C(3)(15)N(1)]-pantothenate after three passages of the murine cells in culture. Charcoal-dextran-stripped fetal bovine serum (FBS) was found to be more efficient for serum supplementation than dialyzed or undialyzed FBS, due to lower contaminating unlabeled pantothenate content. Stable isotope labeled CoA species were extracted and utilized as internal standards for CoA thioester analysis in cell culture models. This methodology of stable isotope labeling by essential nutrients in cell culture (SILEC) can serve as a paradigm for using vitamins and other essential nutrients to generate stable isotope standards that cannot be readily synthesized.     

Enhanced sample multiplexing for nitrotyrosine-modified proteins using combined precursor isotopic labeling and isobaric tagging

Current strategies for identification and quantification of 3-nitrotyrosine (3NT) post-translationally modified proteins (PTM) generally rely on biotin/avidin enrichment. Quantitative approaches have been demonstrated which employ isotopic labeling or isobaric tagging in order to quantify differences in the relative abundances of 3NT-modified proteins in two or potentially eight samples, respectively. Here, we present a novel strategy which uses combined precursor isotopic labeling and isobaric tagging (cPILOT) to increase the multiplexing capability of quantifying 3NT-modified proteins to 12 or 16 samples using commercially available tandem mass tags (TMT) or isobaric tags for relative and absolute quantification (iTRAQ), respectively. This strategy employs "light" and "heavy" labeled acetyl groups to block both N-termini and lysine residues of tryptic peptides. Next, 3NT is reduced to 3-aminotyrosine (3AT) using sodium dithionite followed by derivatization of light and heavy labeled 3AT-peptides with either TMT or iTRAQ multiplex reagents. We demonstrate the proof-of-principle utility of cPILOT with in vitro nitrated bovine serum albumin (BSA) and mouse splenic proteins using TMT(0), TMT(6), and iTRAQ(8) reagents and discuss limitations of the strategy.     

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.     

Stable isotope labeling with amino acids in cell culture based mass spectrometry approach to detect transient protein interactions using substrate trapping

The analysis of protein interactors in protein complexes can yield important insight into protein function and signal transduction. Thus, a reliable approach to distinguish true interactors from nonspecific interacting proteins is of utmost importance for accurate data interpretation. Although stringent purification methods are critical, challenges still remain in the selection of criteria that will permit the objective differentiation of true members of the protein complex from nonspecific background proteins. To address these challenges, we have developed a quantitative proteomic strategy combining stable isotope labeling with amino acids in cell culture (SILAC), affinity substrate trapping, and gel electrophoresis followed by liquid chromatography-tandem mass spectrometry (geLC-MS/MS) protein quantitation. ATP hydrolysis-deficient vacuolar protein sorting-associated protein 4B (Vps4B) was used as the "bait" protein which served as a substrate trap since its lack of ATP hydrolysis enzymatic activity allows the stabilization of its transiently associated interacting proteins. A significant advantage of our approach is the use of our new in-house-developed software program for SILAC-based mass spectrometry quantitation, which further facilitates the differentiation between the bait protein, endogenous bait-interacting proteins, and nonspecific binding proteins based on their protein ratios. The strategy presented herein is applicable to the analysis of other protein complexes whose compositions are dependent upon the ATP hydrolysis activity of the bait protein used in affinity purification studies.     

Top-down approaches for measuring expression ratios of intact yeast proteins using Fourier transform mass spectrometry

The extension of quantitation methods for small peptides to ions above 5 kDa, and eventually to global quantitative proteomics of intact proteins, will require extensive refinement of current analytical approaches. Here we evaluate postgrowth Cys-labeling and 14N/15N metabolic labeling strategies for determination of relative protein expression levels and their posttranslational modifications using top-down mass spectrometry (MS). We show that intact proteins that are differentially alkylated with acrylamide (+71 Da) versus iodoacetamide (+57 Da) have substantial chromatographic shifts during reversed-phase liquid chromatography separation (particularly in peak tails), indicating a requirement for stable isotopes in alkylation tags for top-down MS. In the 14N/15N metabolic labeling strategy, we achieve 98% 15N incorporation in yeast grown 10 generations under aerobic conditions and determine 50 expression ratios using Fourier transform ion cyclotron resonance MS in comparing these cells to anaerobically grown control (14N) cells. We devise quantitative methods for top-down analyses, including a correction factor for accurate protein ratio determination based upon the signal-to-noise ratio. Using a database of 200 yeast protein forms identified previously by top-down MS, we verify the intact mass tag concept for protein identification without tandem MS. Overall, we find that top-down MS promises work flows capable of large-scale proteome profiling using stable isotope labeling and the determination of >5 protein ratios per spectrum.