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.     

Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and 15N-metabolic labeling

We describe the combined use of 15N-metabolic labeling and a cysteine-reactive biotin affinity tag to isolate and quantitate cysteine-containing polypeptides (Cys-polypeptides) from Deinococcus radiodurans as well as from mouse B16 melanoma cells. D. radiodurans were cultured in both natural isotopic abundance and 15N-enriched media. Equal numbers of cells from both cultures were combined and the soluble proteins extracted. This mixture of isotopically distinct proteins was derivatized using a commercially available cysteine-reactive reagent that contains a biotin group. Following trypsin digestion, the resulting modified peptides were isolated using immobilized avidin. The mixture was analyzed by capillary reversed-phase liquid chromatography (LC) online with ion trap mass spectrometry (MS) as well as Fourier transform ion cyclotron resonance (FTICR) MS. The resulting spectra contain numerous pairs of Cyspolypeptides whose mass difference corresponds to the number of nitrogen atoms present in each of the peptides. Designation of Cys-polypeptide pairs is also facilitated by the distinctive isotopic distribution of the 15N-labeled peptides versus their 14N-labeled counterparts. Studies with mouse B16 cells maintained in culture allowed the observation of hundreds of isotopically distinct pairs of peptides by LC-FTICR analysis. The ratios of the areas of the pairs of isotopically distinct peptides showed the expected 1:1 labeling of the 14N and 15N versions of each peptide. An additional benefit from the present strategy is that the 15N-labeled peptides do not display significant isotope-dependent chromatographic shifts from their 14N-labeled counterparts, therefore improving the precision for quantitating peptide abundances. The methodology presented offers an alternate, cost-effective strategy for conducting global, quantitative proteomic measurements.     

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.     

Impact of peptide modifications on the isobaric tags for relative and absolute quantitation method accuracy

In this study, the impact of amino acid modifications on the accuracy of the iTRAQ (isobaric tags for relative and absolute quantitation) method was evaluated. MCF-7 breast cancer cells, cultured in the presence of 17β-estradiol and tamoxifen, were used as a model system. The cells were labeled and analyzed by reversed-phase liquid chromatography and pulsed Q dissociation ion trap tandem mass spectrometry detection. Database searching was performed by using various combinations of amino acid modification allowances, i.e, Lys/Tyr/Cys and amino terminal iTRAQ labeling, Lys methylation, acetylation and carbamylation, and Cys/Met oxidation. Other than the intended Lys/amino terminal iTRAQ labeling, such modifications occur as a result of either enzymatic or sample preparation related reactions and are typically ignored in quantitation analysis to minimize the rate of false-positive peptide identifications. The study revealed that the modifications with the greatest impact on protein identification and quantitation pertain to Lys and Tyr amino acid residues, that by enabling such modifications the number and type of identified proteins will change (by up to 10%), and that the rate of false-positive protein identifications can be maintained below an upper threshold of 5% if appropriate data filtering conditions are used. In addition, the interference of possible posttranslational modifications (i.e., phosphorylation) with iTRAQ quantitation was examined.     

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.     

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.     

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/.     

IsoQuant: a software tool for stable isotope labeling by amino acids in cell culture-based mass spectrometry quantitation

Accurate protein identification and quantitation are critical when interpreting the biological relevance of large-scale shotgun proteomics data sets. Although significant technical advances in peptide and protein identification have been made, accurate quantitation of high-throughput data sets remains a key challenge in mass spectrometry data analysis and is a labor intensive process for many proteomics laboratories. Here, we report a new SILAC-based proteomics quantitation software tool, named IsoQuant, which is used to process high mass accuracy mass spectrometry data. IsoQuant offers a convenient quantitation framework to calculate peptide/protein relative abundance ratios. At the same time, it also includes a visualization platform that permits users to validate the quality of SILAC peptide and protein ratios. The program is written in the C# programming language under the Microsoft .NET framework version 4.0 and has been tested to be compatible with both 32-bit and 64-bit Windows 7. It is freely available to noncommercial users at http://www.proteomeumb.org/MZw.html .     

High-throughput global peptide proteomic analysis by combining stable isotope amino acid labeling and data-dependent multiplexed-MS/MS

In this work, we describe the application of a stable isotope amino acid (lysine) labeling in conjunction with data-dependent multiplexed tandem mass spectrometry (MS/MS) to facilitate the characterization and identification of peptides from proteomic (global protein) digests. Lysine auxotrophic yeast was grown in the presence of 13C-labeled or unlabeled lysine and combined after harvesting in equal proportions. Endoproteinase LysC digestion of the cytosolic fraction produced a global proteomic sample, consisting of heavy/light labeled peptide pairs. Then data-dependent multiplexed-MS/MS was applied to simultaneously select and dissociate only labeled peptide ion pairs. The approach allows differentiation between N-terminal (e.g., b-type ions) and C-terminal fragment ions (e.g., y-type ions) in resulting tandem mass spectra, as well as the capability of differentiation between near-isobaric glutamine and lysine residues. We also describe the utility of peptide composition and fragment information to support peptide identifications and examine the potential application of lysine labeling for differential quantitative protein analysis.     

Software for quantitative proteomic analysis using stable isotope labeling and data independent acquisition

Many software tools have been developed for analyzing stable isotope labeling (SIL)-based quantitative proteomic data using data dependent acquisition (DDA). However, programs for analyzing SIL-based quantitative proteomics data obtained with data independent acquisition (DIA) have yet to be reported. Here, we demonstrated the development of a new software for analyzing SIL data using the DIA method. Performance of the DIA on SYNAPT G2MS was evaluated using SIL-labeled complex proteome mixtures with known heavy/light ratios (H/L = 1:1, 1:5, and 1:10) and compared with the DDA on linear ion trap (LTQ)-Orbitrap MS. The DIA displays relatively high quantitation accuracy for peptides cross all intensity regions, while the DDA shows an intensity dependent distribution of H/L ratios. For the three proteome mixtures, the number of detected SIL-peptide pairs and dynamic range of protein intensities using DIA drop stepwise, whereas no significant changes in these aspects using DDA were observed. The new software was applied to investigate the proteome difference between mouse embryonic fibroblasts (MEFs) and MEF-derived induced pluripotent stem cells (iPSCs) using (16)O/(18)O labeling. Our study expanded the capacities of our UNiquant software pipeline and provided valuable insight into the performance of the two cutting-edge MS platforms for SIL-based quantitative proteomic analysis today.     

Method for quantitative proteomics research by using metal element chelated tags coupled with mass spectrometry

The mass spectrometry-based methods with a stable isotope as the internal standard in quantitative proteomics have been developed quickly in recent years. But the use of some stable isotope reagents is limited by the relative high price and synthetic difficulties. We have developed a new method for quantitative proteomics research by using metal element chelated tags (MECT) coupled with mass spectrometry. The bicyclic anhydride diethylenetriamine-N,N,N',N' ',N' '-pentaacetic acid (DTPA) is covalently coupled to primary amines of peptides, and the ligand is then chelated to the rare earth metals Y and Tb. The tagged peptides are mixed and analyzed by LC-ESI-MS/MS. Peptides are quantified by measuring the relative signal intensities for the Y and Tb tag pairs in MS, which permits the quantitation of the original proteins generating the corresponding peptides. The protein is then identified by the corresponding peptide sequence from its MS/MS spectrum. The MECT method was evaluated by using standard proteins as model sample. The experimental results showed that metal chelate-tagged peptides chromatographically coeluted successfully during the reversed-phase LC analysis. The relative quantitation results were accurate for proteins using MECT. DTPA modification of the N-terminal of peptides promoted cleaner fragmentation (only y-series ions) in mass spectrometry and improved the confidence level of protein identification. The MECT strategy provides a simple, rapid, and economical alternative to current mass tagging technologies available.     

Acid-labile isotope-coded extractants: a class of reagents for quantitative mass spectrometric analysis of complex protein mixtures

Quantitative mass spectrometry using stable isotope-labeled tagging reagents such as isotope-coded affinity tags has emerged as a powerful tool for identification and relative quantitation of proteins in current proteomic studies. Here we describe an integrated approach using both automated two-dimensional liquid chromatography/ mass spectrometry (2D-LC/MS) and a novel class of chemically modified resins, termed acid-labile isotope-coded extractants (ALICE), for quantitative mass spectrometric analysis of protein mixtures. ALICE contains a thiol-reactive group that is used to capture all cysteine (Cys)-containing peptides from peptide mixtures, an acid-labile linker, and a nonbiological polymer. The acid-labile linker is synthesized in both heavy and light isotope-coded forms and therefore enables the direct relative quantitation of peptides/proteins through mass spectrometric analysis. To test the ALICE method for quantitative protein analysis, two model protein mixtures were fully reduced, alkylated, and digested in solution separately and then Cys-containing peptides covalently captured by either light or heavy ALICE. The reacted light and heavy ALICE were mixed and washed extensively under rigorous conditions and the Cys-containing peptides retrieved by mild acid-catalyzed elution. Finally, the eluted peptides were directly subjected to automated 2D-LC/MS for protein identification and LC/MS for accurate relative quantitation. Our initial study showed that quantitation of protein mixtures using ALICE was accurate. In addition, isolation of Cys-containing peptides by the ALICE method was robust and specific and thus yielded very low background in mass spectrometric studies. Overall, the use of ALICE provides improved dynamic range and sensitivity for quantitative mass spectrometric analysis of peptide or protein mixtures.     

In vivo termini amino acid labeling for quantitative proteomics

Quantitative proteomics is one of the research hotspots in the proteomics field and presently maturing rapidly into an important branch. The two most typical quantitative methods, stable isotope labeling with amino acids in cell culture (SILAC) and isobaric tags for relative and absolute quantification (iTRAQ), have been widely and effectively applied in solving various biological and medical problems. Here, we describe a novel quantitative strategy, termed "IVTAL", for in vivo termini amino acid labeling, which combines some advantages of the two methods above. The core of this strategy is a set of heavy amino acid (13)C(6)-arginine and (13)C(6)-lysine and specific endoproteinase Lys-N and Arg-C that yield some labeled isobaric peptides by cell culture and enzymatic digestion, which are indistinguishable in the MS scan but exhibit multiple MS/MS reporter b, y ion pairs in a full mass range that support quantitation. Relative quantification of cell states can be achieved by calculating the intensity ratio of the corresponding reporter b, y ions in the MS/MS scan. The experimental analysis for various proportions of mixed HeLa cell samples indicated that the novel strategy showed an abundance of reliable quantitative information, a high sensitivity, and a good dynamic range of nearly 2 orders of magnitude. IVTAL, as a highly accurate and reliable quantitative proteomic approach, is expected to be compatible with any cell culture system and to be especially effective for the analysis of multiple post-translational modificational sites in one peptide.     

Endoglycosidase-mediated incorporation of 18O into glycans for relative glycan quantitation

Stable isotopic labeling coupled with mass spectrometry analysis is a promising method of detecting quantitative variations in glycans, which may result in aberrant glycosylation in many disorders and diseases. Although various isotopic labeling methods have been used for relative glycan quantitation, enzymatic (18)O labeling, which offers advantages for glycomics similar to those by protease-catalyzed (18)O labeling for proteomics, has not been developed yet. In this study, endoglycosidase incorporated (18)O into the N-glycan reducing end in (18)O-water as N-glycans were released from glycoproteins, rendering glycan reducing-end (18)O labeling (GREOL) a potential strategy for relative glycan quantitation. This proposed method provided good linearity with high reproducibility within 2 orders of magnitude in dynamic range. The ability of GREOL to quantitatively discriminate between isomeric hybrid N-glycans and complex N-glycans in glycoproteins was validated due to the distinct substrate specificities of endoglycosidases. GREOL was also used to analyze changes in human serum N-glycans associated with hepatocellular carcinoma.     

Large-scale unrestricted identification of post-translation modifications using tandem mass spectrometry

TwinPeaks, a close variant of the SEQUEST protein identification algorithm, is capable of unrestricted, large-scale, identification of post-translation modifications (PTMs). TwinPeaks is applied on a sample of 100441 tandem mass spectra from the HUPO Plasma Proteome Project data set, with full non-redundant human as a reference protein database. With a 3.5% error rate, TwinPeaks identifies a collection of 539 spectra that were not identified by the usual PTM-restricted identification algorithm. At this error rate, TwinPeaks increases the rate of spectra identifications by at least 17.6%, making unrestricted PTM identification an integral part of proteomics.     

Method development for metaproteomic analyses of marine biofilms

The large-scale identification and quantitation of proteins via nanoliquid chromatography (LC)-tandem mass spectrometry (MS/MS) offers a unique opportunity to gain unprecedented insight into the microbial composition and biomolecular activity of true environmental samples. However, in order to realize this potential for marine biofilms, new methods of protein extraction must be developed as many compounds naturally present in biofilms are known to interfere with common proteomic manipulations and LC-MS/MS techniques. In this study, we used amino acid analyses (AAA) and LC-MS/MS to compare the efficacy of three sample preparation methods [6 M guanidine hydrochloride (GuHCl) protein extraction + in-solution digestion + 2D LC; sodium dodecyl sulfate (SDS) protein extraction + 1D gel LC; phenol protein extraction + 1D gel LC] for the metaproteomic analyses of an environmental marine biofilm. The AAA demonstrated that proteins constitute 1.24% of the biofilm wet weight and that the compared methods varied in their protein extraction efficiencies (0.85-15.15%). Subsequent LC-MS/MS analyses revealed that the GuHCl method resulted in the greatest number of proteins identified by one or more peptides whereas the phenol method provided the greatest sequence coverage of identified proteins. As expected, metagenomic sequencing of the same biofilm sample enabled the creation of a searchable database that increased the number of protein identifications by 48.7% (≥1 peptide) or 54.7% (≥2 peptides) when compared to SwissProt database identifications. Taken together, our results provide methods and evidence-based recommendations to consider for qualitative or quantitative biofilm metaproteome experimental design.     

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.     

Proteomic complex detection using sedimentation

Protein-protein interactions are important in many cellular processes, but there are still relatively few methods to screen for novel protein complexes. Here we present a quantitative proteomics technique called ProCoDeS (Proteomic Complex Detection using Sedimentation) for profiling the sedimentation of a large number of proteins through a rate zonal centrifugation gradient. Proteins in a putative complex can be identified since they sediment faster than predicted from their monomer molecular weight. Using solubilized mitochondrial membrane proteins from Arabidopsis thaliana, the relative protein abundance in fractions of a rate zonal gradient was measured with the isotopic labeling reagent ICAT and electrospray mass spectrometry. Subunits of the same protein complex had very similar gradient distribution profiles, demonstrating the reproducibility of the quantitation method. The approximate size of the unknown complex can be inferred from its sedimentation rate relative to known protein complexes. ProCoDeS will be of use in screening extracts of tissues, cells, or organelle fractions to identify specific proteins in stable complexes that can be characterized by subsequent targeted techniques such as affinity tagging.