We developed a high-throughput mass spectrometry method, pLink-SS (http://pfind.ict.ac.cn/software/pLink/2014/pLink-SS.html), for precise identification of disulfide-linked peptides. Using pLink-SS, we mapped all native disulfide bonds of a monoclonal antibody and ten standard proteins. We performed disulfide proteome analyses and identified 199 disulfide bonds in Escherichia coli and 568 in proteins secreted by human endothelial cells. We discovered many regulatory disulfide bonds involving catalytic or metal-binding cysteine residues.

Database search programs are essential tools for identifying peptides via mass spectrometry (MS) in shotgun proteomics. Simultaneously achieving high sensitivity and high specificity during a database search is crucial for improving proteome coverage. Here we present JUMP, a new hybrid database search program that generates amino acid tags and ranks peptide spectrum matches (PSMs) by an integrated score from the tags and pattern matching. In a typical run of liquid chromatography coupled with high-resolution tandem MS, more than 95% of MS/MS spectra can generate at least one tag, whereas the remaining spectra are usually too poor to derive genuine PSMs. To enhance search sensitivity, the JUMP program enables the use of tags as short as one amino acid. Using a target-decoy strategy, we compared JUMP with other programs (e.g. SEQUEST, Mascot, PEAKS DB, and InsPecT) in the analysis of multiple datasets and found that JUMP outperformed these preexisting programs. JUMP also permitted the analysis of multiple co-fragmented peptides from "mixture spectra" to further increase PSMs. In addition, JUMP-derived tags allowed partial de novo sequencing and facilitated the unambiguous assignment of modified residues. In summary, JUMP is an effective database search algorithm complementary to current search programs. Molecular & Cellular Proteomics 13: 10.1074/mcp.O114.039586, 3663-3673, 2014.Peptide identification by tandem mass spectra is a critical step in mass spectrometry (MS)-based 1 proteomics (1). Numerous computational algorithms and software tools have been developed for this purpose (2-6). These algorithms can be classified into three categories: (i) pattern-based database search, (ii) de novo sequencing, and (iii) hybrid search that combines database search and de novo sequencing. With the continuous development of high-performance liquid chromatography and high-resolution mass spectrometers, it is now possible to analyze almost all protein components in mammalian cells (7). In contrast to rapid data collection, it remains a challenge to extract accurate information from the raw data to identify peptides with low false positive rates (specificity) and minimal false negatives (sensitivity) (8).Database search methods usually assign peptide sequences by comparing MS/MS spectra to theoretical peptide spectra predicted from a protein database, as exemplified in SEQUEST (9), Mascot (10), OMSSA (11), X!Tandem (12), Spectrum Mill (13), ProteinProspector (14), MyriMatch (15), Crux (16), MS-GFDB (17), Andromeda (18), BaMS 2 (19), and Morpheus (20). Some other programs, such as SpectraST (21) and Pepitome (22), utilize a spectral library composed of experimentally identified and validated MS/MS spectra. These methods use a variety of scoring algorithms to rank potential peptide spectrum matches (PSMs) and select the top hit as a putative PSM. However, not all PSMs are correctly assigned. For example, false peptides may be assigned to MS/MS spectra with numerous noisy peaks and poor fragmentation patterns. If the samples conta...

Proteogenomics is an emerging approach to improve gene annotation and interpretation of proteomics data. Here we present JUMPg, an integrative proteogenomics pipeline including customized database construction, tag-based database search, peptide-spectrum match filtering, and data visualization. JUMPg creates multiple databases of DNA polymorphisms, mutations, splice junctions, partially trypticity, as well as protein fragments translated from the whole transcriptome in all six frames upon RNA-seq de novo assembly. We use a multistage strategy to search these databases sequentially, in which the performance is optimized by researching only unmatched high quality spectra, and re-using amino acid tags generated by the JUMP search engine. The identified peptides/proteins are displayed with gene loci using the UCSC genome browser. Then the JUMPg program is applied to process a label-free mass spectrometry dataset of Alzheimer’s disease postmortem brain, uncovering 496 new peptides of amino acid substitutions, alternative splicing, frame shift, and “non-coding gene” translation. The novel protein PNMA6BL specifically expressed in the brain is highlighted. We also tested JUMPg to analyze a stable-isotope labeled dataset of multiple myeloma cells, revealing 991 sample-specific peptides that include protein sequences in the immunoglobulin light chain variable region. Thus, the JUMPg program is an effective proteogenomics tool for multi-omics data integration.

The mind bomb 1 (Mib1) ubiquitin ligase is essential for controlling metazoan development by Notch signaling and possibly the Wnt pathway. It is also expressed in postmitotic neurons and regulates neuronal morphogenesis and synaptic activity by mechanisms that are largely unknown. We sought to comprehensively characterize the Mib1 interactome and study its potential function in neuron development utilizing a novel sequential elution strategy for affinity purification, in which Mib1 binding proteins were eluted under different stringency and then quantified by the isobaric labeling method. The strategy identified the Mib1 interactome with both deep coverage and the ability to distinguish high-affinity partners from low-affinity partners. A total of 817 proteins were identified during the Mib1 affinity purification, including 56 high-affinity partners and 335 low-affinity partners, whereas the remaining 426 proteins are likely copurified contaminants or extremely weak binding proteins. The analysis detected all previously known Mib1-interacting proteins and revealed a large number of novel components involved in Notch and Wnt pathways, endocytosis and vesicle transport, the ubiquitin-proteasome system, cellular morphogenesis, and synaptic activities. Immunofluorescence studies further showed colocalization of Mib1 with five selected proteins: the Usp9x (FAM) deubiquitinating enzyme, alpha-, beta-, and delta-catenins, and CDKL5. Mutations of CDKL5 are associated with early infantile epileptic encephalopathy-2 (EIEE2), a severe form of mental retardation. We found that the expression of Mib1 downregulated the protein level of CDKL5 by ubiquitination, and antagonized CDKL5 function during the formation of dendritic spines. Thus, the sequential elution strategy enables biochemical characterization of protein interac-

Alzheimer's disease (AD) is the most common form of dementia, characterized by progressive loss of cognitive function. One of the pathological hallmarks of AD is the formation of neurofibrillary tangles composed of abnormally hyperphosphorylated tau protein, but global deregulation of protein phosphorylation in AD is not well analyzed. Here we report a pilot investigation of AD phosphoproteome by titanium dioxide enrichment coupled with high resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). During the optimization of the enrichment method, we found that phosphate ion at a low concentration (e.g. 1 mM) worked efficiently as a non-phosphopeptide competitor to reduce background. The procedure was further tuned with respect to peptide-to-bead ratio, phosphopeptide recovery and purity. Using this refined method and 9 h LC-MS/MS, we analyzed phosphoproteome in one milligram of digested AD brain lysate, identifying 5,243 phosphopeptides containing 3,715 non-redundant phosphosites on 1,455 proteins, including 31 phosphosites on the tau protein. This modified enrichment method is simple and highly efficient. The AD case study demonstrates its feasibility of dissecting phosphoproteome in a limited amount of postmortem human brain.

Alternative polyadenylation (APA) plays an important role in gene expression by affecting mRNA stability, translation, and translocation in cells. However, genome-wide APA events have only recently been subjected to more systematic analysis with newly developed high-throughput methods. In this review, we focus on the recent technological development of APA analyses on a genome-wide scale, as well as the impact of APA switches on a number of critical biological processes in animals, including cell proliferation, differentiation, and oncogenic transformation. With the highly enlarged scope of genome-wide APA analyses, the APA regulations of various biological processes have increasingly become a new paradigm for the regulation of gene transcription and translation.

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