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The advent of high-resolution and frequency mass spectrometry has ushered in an era of data-independent acquisition (DIA). This approach affords enormous multiplexing capacity and is particularly suitable for clinical biomarker studies. However, DIA-based quantification of clinical plasma samples is a daunting task due to the high complexity of clinical plasma samples, the diversity of peptides within the samples, and the high biologic dynamic range of plasma proteins. Here we applied DIA methodology, including a highly reproducible sample preparation and LC-MS/MS analysis, and assessed its utility for clinical plasma biomarker detection. A pancreatic cancer-relevant plasma spectral library was constructed consisting of over 14 000 confidently identified peptides derived from over 2300 plasma proteins. Using a nonhuman protein as the internal standard, various empirical parameters were explored to maximize the reliability and reproducibility of the DIA quantification. The DIA parameters were optimized based on the quantification cycle times and fragmentation profile complexity. Higher analytical and biological reproducibility was recorded for the tryptic peptides without labile residues and missed cleavages. Quantification reliability was developed for the peptides identified within a consistent retention time and signal intensity. Linear analytical dynamic range and the lower limit of quantification were assessed, suggesting the critical role of sample complexity in optimizing DIA settings. Technical validation of the assay using a cohort of clinical plasma indicated the robustness and unique advantage for targeted analysis of clinical plasma samples in the context of biomarker development.

High-density lipoprotein (HDL), a lipid nanoparticle containing many different low abundance proteins, is an attractive target for clinical proteomics because its compositional heterogeneity is linked to its cardioprotective effects. Selected reaction monitoring (SRM) is currently the method of choice for targeted quantification of proteins in such a complex biological matrix. However, model system studies suggest that parallel reaction monitoring (PRM) is more specific than SRM because many product ions can be used to confirm the identity of a peptide. We therefore compared PRM and SRM for their abilities to quantify proteins in HDL, using (15)N-labeled apolipoprotein A-I (HDL's most abundant protein) as the internal standard. PRM and SRM exhibited comparable linearity, dynamic range, precision, and repeatability for protein quantification of HDL. Moreover, the single internal standard protein performed as well as protein-specific peptide internal standards when quantifying 3 different proteins. Importantly, PRM and SRM yielded virtually identical quantitative results for 26 proteins in HDL isolated from 44 subjects. Because PRM requires less method development than SRM and is potentially more specific, our observations indicate that PRM in concert with a single isotope-labeled protein is a promising new strategy for quantifying HDL proteins in translational studies., Biological Significance: HDL, a complex matrix composed of lipids and proteins, is implicated in cardioprotection. Its cholesterol content correlates inversely with cardiovascular disease and it is the current metric to assess cardiovascular risk. However, the cholesterol content does not capture HDL's complexity and heterogeneity. Devising metrics that better capture HDL's cardioprotective effects, we developed an optimized method for quantification of HDL proteome, using PRM in concert with a single labeled protein as internal standard. The availability of a method that increases sample throughput without compromising the reproducibility, sensitivity, and accuracy could therefore point to better risk assessment for CVD or other diseases., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Advances in phosphoproteomics have made it possible to monitor changes in protein phosphorylation that occur at different steps in signal transduction and have aided the identification of new pathway components. In the present study, we applied this technology to advance our understanding of the responses of melanoma cells to signaling initiated by the secreted ligand WNT3A. We started by comparing the phosphopeptide patterns of cells treated with WNT3A for different periods of time. Next, we integrated these data sets with the results from a siRNA screen that targeted protein kinases. This integration of siRNA screening and proteomics enabled us to identify four kinases that exhibit altered phosphorylation in response to WNT3A and that regulate a luciferase reporter of β-catenin-responsive transcription (β-catenin-activated reporter). We focused on one of these kinases, an atypical PKC kinase, protein kinase N1 (PKN1). Reducing the levels of PKN1 with siRNAs significantly enhances activation of β-catenin-activated reporter and increases apoptosis in melanoma cell lines. Using affinity purification followed by mass spectrometry, we then found that PKN1 is present in a protein complex with a WNT3A receptor, Frizzled 7, as well as with proteins that co-purify with Frizzled 7. These data establish that the protein kinase PKN1 inhibits Wnt/β-catenin signaling and sensitizes melanoma cells to cell death stimulated by WNT3A.

Cellular control of protein activities by modulation of their abundance or compartmentalization is not easily measured on a large scale. We developed and applied a method to globally interrogate these processes that is widely useful for systems-level analyses of dynamic cellular responses in many cell types. The approach involves subcellular fractionation followed by comprehensive proteomic analysis of the fractions, which is enabled by a data-independent acquisition mass spectrometry approach that samples every available mass to charge channel systematically to maximize sensitivity. Next, various fraction-enrichment ratios are measured for all detected proteins across different environmental conditions and used to group proteins into clusters reflecting changes in compartmentalization and relative conditional abundance. Application of the approach to characterize the response of yeast proteins to fatty acid exposure revealed dynamics of peroxisomes and novel dynamics of MCC/eisosomes, specialized plasma membrane domains comprised of membrane compartment occupied by Can1 (MCC) and eisosome subdomains. It also led to the identification of Fat3, a fatty acid transport protein of the plasma membrane, previously annotated as Ykl187.

Sites of ubiquitin modification have been identified by mass spectrometry based on the increase in molecular mass of a tryptic peptide carrying two additional glycine residues from the ubiquitin moiety. However, such peptides with GG shifts have been difficult to discover. We identify 870 unique sites of ubiquitin attachment on 438 different proteins of the yeast Saccharomyces cerevisiae., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The 1310 Haloarcula marismortui proteins identified from mid-log and late-log phase soluble and membrane proteomes were analyzed in metabolic and cellular process networks to predict the available systems and systems fluctuations upon environmental stresses. When the connected metabolic reactions with identified proteins were examined, the availability of a number of metabolic pathways and a highly connected amino acid metabolic network were revealed. Quantitative spectral count analyses suggested 300 or more proteins might have expression changes in late-log phase. Among these, integrative network analyses indicated approximately 106 were metabolic proteins that might have growth-phase dependent changes. Interestingly, a large proportion of proteins in affected biomodules had the same trend of changes in spectral counts. Disregard the magnitude of changes, we had successfully predicted and validated the expression changes of nine genes including the rimK, gltCP, rrnAC0132, and argC in lysine biosynthesis pathway which were downregulated in late-log phase. This study had not only revealed the expressed proteins but also the availability of biological systems in two growth phases, systems level changes in response to the stresses in late-log phase, cellular locations of identified proteins, and the likely regulated genes to facilitate further analyses in the postgenomic era.

The Halobacterium salinarum gas vesicle (GV) is an extremely stable intracellular organelle with air trapped inside a proteinaceous membrane. Reported here is a comparative proteomics analysis of GV and GV depleted lysate (GVD) to reveal the membrane structural proteins. Ten proteins encoded by gvp-1 (gvpMLKJIHGFED-1 and gvpACNO-1) and five proteins encoded by gvp-2 (gvpMLKJIHGFED-2 and gvpACNO-2) gene clusters for the biogenesis of spindle- and cylindrical-, respectively, shaped GV were identified by LC-MS/MS. The peptides of GvpA1, I1, J1, A2, and J2 were exclusively identified in purified GV, GvpD1, H1, L1, and F2 only in GVD, and GvpC1, N1, O1, F1, H2, and O2 in both samples. The identification of GvpA1, C1, F1, J1, and A2 in GV is in agreement with their previously known structural function. In addition, the detection of GvpI1, N1, O1, H2, J2, and O2 in GV suggested they are new structural proteins. Among these, the structural role of GvpI1 and N1 in GV was further validated by immuno-detection of protein A-tagged GvpI1 and N1 fusion proteins in purified GV. Thus, LC-MS/MS could reveal at least a half dozen gas vesicle structural proteins in the predominant spindle-shaped GV that may be helpful for studying its biogenesis.

Electron-transfer dissociation (ETD) induces fragmentation along the peptide backbone by transferring an electron from a radical anion to a protonated peptide. In contrast with collision-induced dissociation, side chains and modifications such as phosphorylation are left intact through the ETD process. Because the precursor charge state is an important input to MS/MS sequence database search tools, the ability to accurately determine the precursor charge is helpful for the identification process. Furthermore, because ETD can be applied to large, highly charged peptides, the need for accurate precursor charge state determination is magnified. Otherwise, each spectrum must be searched repeatedly using a large range of possible precursor charge states. To address this problem, we have developed an ETD charge state prediction tool based on support vector machine classifiers that is demonstrated to exhibit superior classification accuracy while minimizing the overall number of predicted charge states. The tool is freely available, open source, cross platform compatible, and demonstrated to perform well when compared with an existing charge state prediction tool. The program is available from http://code.google.com/p/etdz/.

A by-product in the processing of prostate tissue for cell sorting by collagenase digestion is the media supernatant that remains after the cells are harvested. These supernatants contain proteins made by the cells within the tissue. Quantitative proteomic analysis of N-glycosylated proteins detected an increased amount of CD90/THY1 in cancer supernatants compared with non-cancer supernatants. Immunohistochemistry showed that in all carcinomas, regardless of Gleason grade, a layer of CD90-positive stromal fibroblastic cells, ∼5 to 10 cells deep, was localized to tumor glands. In contrast, a no more than 1-cell wide girth of CD90-positive stromal cells was found around benign glands. The increased number of CD90-positive stromal cells in cancer correlated with overexpression of CD90 mRNA detected by gene expression analysis of stromal cells obtained by laser-capture microdissection. There is increasing evidence that cancer-associated stroma has a function in both tumor progression and carcinogenesis. Most experiments to identify cancer biomarkers have focused on the cancer cells. CD90, being a marker for prostate cancer-associated stroma, might be a potential biomarker for this cancer. A non-invasive test could be provided by a urine test. Proteomic analysis of urine from patients with prostate cancer identified CD90; conversely, CD90 was not detected in the urine of post-prostatectomy patients. Furthermore, this urinary CD90 protein was a variant CD90 protein not known to be expressed by such cells as lymphocytes that express CD90. These CD90 results were obtained from ∼90 cases consisting of proteomic analysis of tissue and urine, immunohistochemistry, western blot analysis of tissue media, flow cytometry of cells from digested tissue, and reverse transcriptase polymerase chain reaction analysis of isolated stromal cells.

MS-based proteomics characterizes protein contents of biological samples. The most common approach is to first match observed MS/MS peptide spectra against theoretical spectra from a protein sequence database and then to score these matches. The false discovery rate (FDR) can be estimated as a function of the score by searching together the protein sequence database and its randomized version and comparing the score distributions of the randomized versus nonrandomized matches. This work introduces a straightforward isotonic regression-based method to estimate the cumulative FDRs and local FDRs (LFDRs) of peptide identification. Our isotonic method not only performed as well as other methods used for comparison, but also has the advantages of being: (i) monotonic in the score, (ii) computationally simple, and (iii) not dependent on assumptions about score distributions. We demonstrate the flexibility of our approach by using it to estimate FDRs and LFDRs for protein identification using summaries of the peptide spectra scores. We reconfirmed that several of these methods were superior to a two-peptide rule. Finally, by estimating both the FDRs and LFDRs, we showed for both peptide and protein identification, moderate FDR values (5%) corresponded to large LFDR values (53 and 60%).

In this paper, we present the results of proof-of-concept experiments using a novel photocleavable and mass spectrometry identifiable cross-linker pcPIR (photocleavable protein interaction reporter). pcPIR can be dissociated under UV irradiation either off- or online before the introduction to the mass spectrometers. Photo dissociation of cross-linkers is different from either the gas phase or the chemical cleavage of cross-linkers. Different types of cross-links can be identified using the pcPIR mass relationships, where the mass of cross-linked precursor equals the sum of the masses of the released products and reporter. Since pcPIR is cleaved prior to the entrance to the mass spectrometer, the released peptides are available to be sequenced with routine collision-induced dissociation (CID) MS/MS experiments and database search algorithms. In this report, the pcPIR strategy of identifying the cross-linked peptides with on- and off-line photocleavage coupled with novel targeted data dependent LC-MS/MS is demonstrated with the use of standard peptides, bovine serum albumin (BSA), and human hemoglobin tetramer protein complex.

Electron transfer dissociation (ETD) is an alternative fragmentation technique to CID that has recently become commercially available. ETD has several advantages over CID. It is less prone to fragmenting amino acid side chains, especially those that are modified, thus yielding fragment ion spectra with more uniform peak intensities. Further, precursor ions of longer peptides and higher charge states can be fragmented and identified. However, analysis of ETD spectra has a few important differences that require the optimization of the software packages used for the analysis of CID data or the development of specialized tools. We have adapted the Trans-Proteomic Pipeline to process ETD data. Specifically, we have added support for fragment ion spectra from high-charge precursors, compatibility with charge-state estimation algorithms, provisions for the use of the Lys-C protease, capabilities for ETD spectrum library building, and updates to the data formats to differentiate CID and ETD spectra. We show the results of processing data sets from several different types of ETD instruments and demonstrate that application of the ETD-enhanced Trans-Proteomic Pipeline can increase the number of spectrum identifications at a fixed false discovery rate by as much as 100% over native output from a single sequence search engine.

Data-dependent precursor ion selection is widely used in shotgun proteomics to profile the protein components of complex samples. Although very popular, this bottom-up method presents major drawbacks in terms of detectable dynamic range. Here, we demonstrate the superior performance of a data-independent method we term precursor acquisition independent from ion count (PAcIFIC). Our results show that almost the entire, predicted, soluble bacterial proteome can be thoroughly analyzed by PAcIFIC without the need for any sample fractionation other than the C18-based liquid chromatograph used to introduce the peptide mixture into the mass spectrometer. Importantly, we also show that PAcIFIC provides unique performance for analysis of human plasma in terms of the number of proteins identified (746 at FDR < or = 0.5%) and achieved dynamic range (8 orders of magnitude at FDR < or = 0.5%), without any fractionation other than immuno-depletion of the seven most abundant proteins.

The identification of peptides by microcapillary liquid chromatography-tandem mass spectrometry (microLC-MS/MS) has become routine because of the development of fast scanning mass spectrometers, data-dependent acquisition, and database searching algorithms. However, many peptides within the detection limit of the mass spectrometer remain unidentified because of limitations in MS/MS sampling speed despite the dynamic range and peak capacity of the instrument. We have developed an automated approach that uses the mass spectra from high resolution microLC-MS data to define the molecular species present in the mixture and directs the acquisition of MS/MS spectra to precursors that were missed in prior analyses. This approach increases the coverage of the molecular species sampled by MS/MS and consequently the number of peptides and proteins identified during the acquisition of technical or biological replicates using a simple one-dimensional chromatographic separation. The combination of a unique workflow and custom software contribute to the improved identification of molecular features detected in proteomics experiments of complex protein mixtures.

The identification and characterization of previously unidentified signal transduction molecules has expanded our understanding of biological systems and facilitated the development of mechanism-based therapeutics. We present a highly validated small interfering RNA (siRNA) screen that functionally annotates the human genome for modulation of the Wnt/beta-catenin signal transduction pathway. Merging these functional data with an extensive Wnt/beta-catenin protein interaction network produces an integrated physical and functional map of the pathway. The power of this approach is illustrated by the positioning of siRNA screen hits into discrete physical complexes of proteins. Similarly, this approach allows one to filter discoveries made through protein-protein interaction screens for functional contribution to the phenotype of interest. Using this methodology, we characterized AGGF1 as a nuclear chromatin-associated protein that participates in beta-catenin-mediated transcription in human colon cancer cells.

This review focuses on how membrane lipid rafts have been detected and isolated, mostly from lymphocytes, and their associated proteins identified. These proteins include transmembrane antigens/receptors, GPI-anchored proteins, cytoskeletal proteins, Src-family protein kinases, G-proteins, and other proteins involved in signal transduction. To further understand the biology of lipid rafts, new methodological approaches are needed to help characterize the raft protein component, and changes that occur in this component as a result of cell perturbation. We describe the application of new proteomic approaches to the identification and quantification of raft proteins in T-lymphocytes. Similar approaches, applied to other model cell systems, will provide valuable new insights into both cellular signal transduction and lipid raft biology.

Proteomic approaches to biological research that will prove the most useful and productive require robust, sensitive, and reproducible technologies for both the qualitative and quantitative analysis of complex protein mixtures. Here we applied the isotope-coded affinity tag (ICAT) approach to quantitative protein profiling, in this case proteins that copurified with lipid raft plasma membrane domains isolated from control and stimulated Jurkat human T cells. With the ICAT approach, cysteine residues of the two related protein isolates were covalently labeled with isotopically normal and heavy versions of the same reagent, respectively. Following proteolytic cleavage of combined labeled proteins, peptides were fractionated by multidimensional chromatography and subsequently analyzed via automated tandem mass spectrometry. Individual tandem mass spectrometry spectra were searched against a human sequence database, and a variety of recently developed, publicly available software applications were used to sort, filter, analyze, and compare the results of two repetitions of the same experiment. In particular, robust statistical modeling algorithms were used to assign measures of confidence to both peptide sequences and the proteins from which they were likely derived, identified via the database searches. We show that by applying such statistical tools to the identification of T cell lipid raft-associated proteins, we were able to estimate the accuracy of peptide and protein identifications made. These tools also allow for determination of the false positive rate as a function of user-defined data filtering parameters, thus giving the user significant control over and information about the final output of large-scale proteomic experiments. With the ability to assign probabilities to all identifications, the need for manual verification of results is substantially reduced, thus making the rapid evaluation of large proteomic datasets possible. Finally, by repeating the experiment, information relating to the general reproducibility and validity of this approach to large-scale proteomic analyses was also obtained.

Lipid rafts were prepared according to standard protocols from Jurkat T cells stimulated via T cell receptor/CD28 cross-linking and from control (unstimulated) cells. Co-isolating proteins from the control and stimulated cell preparations were labeled with isotopically normal (d0) and heavy (d8) versions of the same isotope-coded affinity tag (ICAT) reagent, respectively. Samples were combined, proteolyzed, and resultant peptides fractionated via cation exchange chromatography. Cysteine-containing (ICAT-labeled) peptides were recovered via the biotin tag component of the ICAT reagents by avidin-affinity chromatography. On-line micro-capillary liquid chromatography tandem mass spectrometry was performed on both avidin-affinity (ICAT-labeled) and flow-through (unlabeled) fractions. Initial peptide sequence identification was by searching recorded tandem mass spectrometry spectra against a human sequence data base using SEQUEST software. New statistical data modeling algorithms were then applied to the SEQUEST search results. These allowed for discrimination between likely "correct" and "incorrect" peptide assignments, and from these the inferred proteins that they collectively represented, by calculating estimated probabilities that each peptide assignment and subsequent protein identification was a member of the "correct" population. For convenience, the resultant lists of peptide sequences assigned and the proteins to which they corresponded were filtered at an arbitrarily set cut-off of 0.5 (i.e. 50% likely to be "correct") and above and compiled into two separate datasets. In total, these data sets contained 7667 individual peptide identifications, which represented 2669 unique peptide sequences, corresponding to 685 proteins and related protein groups.

Bim is a pro-apoptotic member of the Bcl-2 protein family. Bim has three isoforms, EL, L, and S, of which the EL form is the least cytotoxic. We show here that Bim is serine phosphorylated in lymphocytes, predominantly on the EL form. Withdrawal of IL-2 from IL-2-dependent T lymphocytes or culture of thymocytes leads to reduced Bim phosphorylation and apoptosis induction. This decrease in Bim phosphorylation occurs when most cells in culture are still viable, indicating that reduction of Bim phosphorylation may be an early event in apoptosis signaling of lymphocytes.

The two-domain fragment N+K1 (rNK1) [Glu(1)-Glu(163)] of human plasminogen was expressed in E. coli as a hexahistidine-tagged fusion protein and chromatographically purified. The (1)H NMR spectrum supports proper folding of the K1 component within the refolded rNK1 construct (rNK1/K1). The functional properties of rNK1/K1 were investigated via intrinsic fluorescence titration with kringle-specific omega-aminocarboxylic acid ligands. The affinities closely match those previously measured for the isolated K1, which indicates that the N-domain does not significantly affect the interaction of ligands with the lysine binding site of K1. Far-UV CD spectra recorded for the N-domain suggest conformational plasticity and flexibility for the module. Two classes of spectra, referred to as types A and B, were identified with the type A spectrum reflecting a higher secondary structure content than that estimated for the type B spectrum. Subtracting the CD spectrum of rK1 from that of rNK1 yields a spectrum (Delta) which reflects the conformation of the N-domain within the rNK1 construct (rNK1/N). Delta resembles the type A spectrum, suggesting that rNK1/N adopts a relatively more ordered conformation, stabilized by the adjacent rNK1/K1 domain. In contrast, thermal unfolding curves determined via CD indicate that the rNK1/N slightly lowers the melting temperature (T(m)) of rNK1/K1. Independence of the two domains within rNK1 was tested by monitoring the thermal unfolding of rNK1/K1 when in the presence of the kringle-specific ligand AMCHA, which left the rNK1/N T(m) essentially unaffected, while increasing that of the rNK1/K1 by approximately 10 degrees C.

Plasma membranes of most cell types are thought to contain microdomains commonly referred to as lipid rafts, biochemically distinct from bulk plasma membrane, apparently enriched for proteins involved in signal transduction. In T cells, it is believed that lipid rafts aggregate at the site of T cell receptor engagement and act as foci for initiation of the signaling process. In order to gain insight into the possible functioning of lipid rafts, we applied microcapillary liquid chromatography electrospray ionization tandem mass spectrometry (microLC-ESI-MS/MS) methodologies to the identification of proteins which copurified with lipid rafts. Following isolation of lipid rafts as Triton-insoluble, low-density membrane fractions from Jurkat T cells, tryptic digests were generated of individual protein bands resolved electrophoretically. Alternatively, cysteine-containing peptides were isolated from total tryptic digests of unseparated lipid raft proteins following labeling with a cysteine-specific biotinylation reagent and avidin affinity purification. In both cases, protein identifications were made by comparison of tandem MS spectra generated by microLC-ESI-MS/MS to both protein and DNA sequence databases using Sequest software. Proteins identified essentially fell into two groups: cytoskeletal proteins, and proteins involved in signal transduction. These findings are discussed in the light of the current understanding of both lipid raft biology and signal transduction.