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Protein acylation plays a critical role in protein localization and function. Acylation is essential for human immunodeficiency virus 1 (HIV-1) assembly and budding of HIV-1 from the plasma membrane in lipid raft microdomains and is mediated by myristoylation of the Gag polyprotein and the copackaging of the envelope protein is facilitated by colocalization mediated by palmitoylation. Since the viral accessory protein NEF has been shown to alter the substrate specificity of myristoyl transferases, and alter cargo trafficking lipid rafts, we hypothesized that HIV-1 infection may alter protein acylation globally. To test this hypothesis, we labeled HIV-1 infected cells with biomimetics of acyl azides, which are incorporated in a manner analogous to natural acyl-Co-A. A terminal azide group allowed us to use a copper catalyzed click chemistry to conjugate the incorporated modifications to a number of substrates to carry out SDS-PAGE, fluorescence microscopy, and enrichment for LC-MS/MS. Using LC-MS/MS, we identified 103 and 174 proteins from the myristic and palmitic azide enrichments, with 27 and 45 proteins respectively that differentiated HIV-1 infected from uninfected cells. This approach has provided us with important insights into HIV-1 biology and is widely applicable to many virological systems., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: The identification of protein isoforms in complex biological samples is challenging. We, therefore, used an MS approach to unambiguously identify cardiac myofilament protein isoforms based on the observation of a tryptic peptide consisting of a sequence unique to a particular isoform., Experimental Design: Three different workflows were used to isolate and fractionate rat cardiac myofilament subproteomes. All fractions were analyzed on an LTQ-Orbitrap MS, proteins were identified using various search engines (MASCOT, X!Tandem, X!Tandem Kscore, and OMSSA) with results combined via PepArML Meta-Search engine, and a postsearch analysis was performed by MASPECTRAS. All MS data have been deposited in the ProteomeXchange with identifier PXD000874 (http://proteomecentral.proteomexchange.org/dataset/PXD000874)., Results: The combination of multiple workflows and search engines resulted in a larger number of nonredundant proteins identified than with individual methods. A total of 102 myofilament annotated proteins were observed overlapping in two or three of the workflows. Literature search for myofilament presence with manual validation of the MS spectra was carried out for unambiguous identification: ten cardiac myofilament and 17 cardiac myofilament-associated proteins were identified with 39 isoforms and subisoforms., Conclusion and Clinical Relevance: We have identified multiple isoforms of myofilament proteins that are present in cardiac tissue using unique tryptic peptides. Changes in distribution of these protein isoforms under pathological conditions could ultimately allow for clinical diagnostics or as therapeutic targets., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

With the proliferation of search engines for the analysis of MS data, multisearch techniques aimed at boosting the discriminating power of the search engines' score functions have recently become popular. Much statistical and algorithmic work has been done, therefore, in order to be able to combine and parse multiple search streams. However, multisearch techniques suffer from long run times, and may have little impact on false negatives because of similar peptide filtering heuristics between searches. This review focuses, rather, on multipass techniques, which use the results of one search to guide the selection of spectra, parameters and sequences in subsequent searches. This reduces the number of false-negative peptide identifications due to peptide candidate filtering while preserving statistical significance of existing (correct) identifications. Furthermore, this technique avoids substantial increases in running time and, by limiting the search space, does not reduce the statistical significance of correct identifications or introduce a statistically significant number of false-positive identifications. However, we argue that the existing combiner tools are not reliably applicable to these multipass situations, because of algorithmic assumptions about search space and statistical assumptions about the rate of true positives. Here we provide an overview of the advantages of and issues in multipass analysis techniques, the existing methods and workflows available to proteomic researchers, and the unsolved statistical and algorithmic issues amenable to future research.

Two-dimensional gel electrophoresis (2-DE) is widely used for initial protein separation in proteomics. Commercial products using neutral pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis ((SDS-PAGE)/(Bis (2-hydroxyethyl) imino-tris (hydroxymethyl) methane-HCl, or Bis-Tris)) have greatly improved this technique, but cost and limited sizes restrict their applications. An "in-house" system is presented, resulting in better resolution, separation, and new spot visualization and improved resolution when compared to Tris-HCl gels. Their utility is demonstrated using albumin-depleted serum samples, rabbit heart left ventricle, and human immunodeficiency virus type 1 (HIV-1).

Proteomics, the study of the proteome (the collection of all the proteins expressed from the genome in all isoforms, polymorphisms and post-translational modifications), is a rapidly developing field in which there are numerous new and often expensive technologies, making it imperative to use the most appropriate technology for the biological system and hypothesis being addressed. This review provides some guidelines on approaching a broad-based proteomics project, including strategies on refining hypotheses, choosing models and proteomic approaches with an emphasis on aspects of sample complexity (including abundance and protein characteristics), and separation technologies and their respective strengths and weaknesses. Finally, issues related to quantification, mass spectrometry and informatics strategies are discussed. The goal of this review is therefore twofold: the first section provides a brief outline of proteomic technologies, specifically with respect to their applications to broad-based proteomic approaches, and the second part provides more details about the application of these technologies in typical scenarios dealing with physiological and pathological processes. Proteomics at its best is the integration of carefully planned research and complementary techniques with the advantages of powerful discovery technologies that has the potential to make substantial contributions to the understanding of disease and disease processes.

Lipidomics, the systematic study of the lipid composition of a cell or tissue, is an invaluable complement to knowledge gained by genomics and proteomics research. Mass spectrometry provides a means to detect hundreds of lipids in parallel, and this includes low abundance species of lipids. Nevertheless, frequently occurring isobaric and isomeric lipid species complicate lipidomics analyses from an analytical and bioinformatics perspective. Various MS/MS strategies have evolved to resolve ambiguous identifications of lipid species, and these strategies have been supported by corresponding bioinformatics analysis tools. This review intends to familiarize readers with available bioinformatics MS/MS analysis tools and databases, the structural information obtainable from these, and their applicability to different MS/MS strategies. Finally, future challenges in detecting double bond positions are investigated from a bioinformatics perspective. [ABSTRACT FROM PUBLISHER]

Abstract: With today''s understanding of the genome, advanced molecular techniques, and our growing ability to analyze complex biologic data, proteomics, the broad-based study of the repertoire of proteins expressed by cells or organisms, offers great promise for understanding the causes of complex diseases. Identifying and measuring novel protein biomarkers present in blood that predict the onset and resolution of laminitis would both aid clinical management of at-risk equine patients and shed light on underlying mechanisms with the intent of developing novel preventive strategies and therapeutic approaches. Identifying alterations in the profiles of proteins present in tissues of horses that develop laminitis or within in vitro model systems also can advance our understanding of this puzzling disease. [Copyright &y& Elsevier]