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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.)

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.

The glycosylation state of envelope glycoproteins in human and simian immunodeficiency viruses (HIV/SIV) is critical to viral infectivity and tropism, viral protein processing, and in virus evasion of the immune system. Using a rapid fluorescent 2-D gel-based method coupled with enzymatic pre-treatment of virus with PNGase F (Peptide: N-Glycosidase F) and fluorescent 2-D gels or 2-D gel Western blotting, we show significant differences in the glycosylation patterns of two SIV strains widely used in animal models of HIV disease and vaccine studies. We also demonstrate the modification of a host protein important in HIV biology (HLA-DR) by O-GlcNAc. Further, this experimental pipeline allows for the identification of the modified protein and the site of N-linked glycosylation by fluorescent 2-DE coupled with MS and the qualitative and semi-quantitative assessment of viral glycosylation. The method is fully compatible with downstream glycomics analysis. This approach will permit correlation of virus glycosylation status with pathological severity and may serve as a rapid screen of viruses from physiological samples for further study by more advanced MS methodology.

We here present a user-friendly and extremely lightweight tool that can serve as a stand-alone front-end for the Open MS Search Algorithm (OMSSA) search engine, or that can directly be used as part of an informatics processing pipeline for MS driven proteomics. The OMSSA graphical user interface (OMSSAGUI) tool is written in Java, and is supported on Windows, Linux, and OSX platforms. It is an open source under the Apache 2 license and can be downloaded from http://code.google.com/p/mass-spec-gui/.

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).