A mass-tolerant database search identifies a large proportion of unassigned spectra in shotgun proteomics as modified peptides

Chick, Joel M.; Kolippakkam, Deepak; Nusinow, David P.; Zhai, Bo; Rad, Ramin; Huttlin, Edward L.; Gygi, Steven P.

doi:10.1038/nbt.3267

Cited by 382 publications

(522 citation statements)

References 43 publications

(49 reference statements)

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“…The high confidence provided from searching with fragment ions measured at accurate mass (Ͻ10 ppm) allows for wide precursor tolerances and "delta M" mode, making it possible to identify proteins with unannotated sequence variations and PTMs. However, proteins that do not have established cleavage sites (proteolytic processing) or that are mis-annotated cannot be identified using this search type unless a large MS 1 tolerance is used in an "open" and error-tolerant search strategy (56). All different branches in the search tree with distinct MS 1 tolerances combined with the deep manual analysis of the obtained identifications allowed us to annotate the 53 unique proteoforms presented here.…”

Section: Discussionmentioning

confidence: 99%

Mapping Proteoforms and Protein Complexes From King Cobra Venom Using Both Denaturing and Native Top-down Proteomics

Melani¹,

Skinner

Fornelli

et al. 2016

Molecular & Cellular Proteomics

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Characterizing whole proteins by top-down proteomics avoids a step of inference encountered in the dominant bottom-up methodology when peptides are assembled computationally into proteins for identification. The direct interrogation of whole proteins and protein complexes from the venom of Ophiophagus hannah (king cobra) provides a sharply clarified view of toxin sequence variation, transit peptide cleavage sites and post-translational modifications (PTMs) likely critical for venom lethality. A tube-gel format for electrophoresis (called GELFrEE) and solution isoelectric focusing were used for protein fractionation prior to LC-MS/MS analysis resulting in 131 protein identifications (18 more than bottom-up) and a total of 184 proteoforms characterized from 14 protein toxin families. Operating both GELFrEE and mass spectrometry to preserve non-covalent interactions generated detailed information about two of the largest venom glycoprotein complexes: the homodimeric L-amino acid oxidase (ϳ130 kDa) and the multichain toxin cobra venom factor (ϳ147 kDa). The L-amino acid oxidase complex exhibited two clusters of multiproteoform complexes corresponding to the presence of 5 or 6 N-glycans moieties, each consistent with a distribution of N-acetyl hexosamines. Employing top-down proteomics in both native and denaturing modes provides unprecedented characterization of venom proteoforms and their complexes. A precise molecular inventory of venom proteins will propel the study of snake toxin variation and the targeted development of new antivenoms or other biotherapeutics. Molecular &

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Section: Discussionmentioning

confidence: 99%

Mapping Proteoforms and Protein Complexes From King Cobra Venom Using Both Denaturing and Native Top-down Proteomics

Melani¹,

Skinner

Fornelli

et al. 2016

Molecular & Cellular Proteomics

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“…It is also possible to repurpose existing datasets to look for PTMs that were not considered in the original analysis, for example via mass‐tolerant database searches 107. This task is made difficult by the substoichiometric nature of modified proteins, thus usually requiring experimental enrichment techniques to enable detection 108, 109, 110.…”

Section: Introductionmentioning

confidence: 99%

Exploring the potential of public proteomics data

et al. 2015

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In a global effort for scientific transparency, it has become feasible and good practice to share experimental data supporting novel findings. Consequently, the amount of publicly available MS‐based proteomics data has grown substantially in recent years. With some notable exceptions, this extensive material has however largely been left untouched. The time has now come for the proteomics community to utilize this potential gold mine for new discoveries, and uncover its untapped potential. In this review, we provide a brief history of the sharing of proteomics data, showing ways in which publicly available proteomics data are already being (re‐)used, and outline potential future opportunities based on four different usage types: use, reuse, reprocess, and repurpose. We thus aim to assist the proteomics community in stepping up to the challenge, and to make the most of the rapidly increasing amount of public proteomics data.

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“…The sequencing of the human genome was considered complete around a decade ago, the human proteome is however still under construction -an almost overwhelming task tolerant search a large portion of these spectra can be assigned to peptides modified by "unusual" modifications [142]. The need for this type of searches is obvious and as computational power continues to grow, so will the field of proteomics.…”

Section: Challenges Of a Proteomic Approachmentioning

confidence: 99%

Human Adipocytes : Proteomic Approaches

Jufvas¹

2016

Linköping University Medical Dissertations

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Type 2 diabetes is characterized by increased levels of glucose in the blood originating from insulin resistance in insulin sensitive tissues and from reduced pancreatic insulin production. Around 400 million people in the world are diagnosed with type 2 diabetes and the correlation with obesity is strong. In addition to life style induction of obesity and type 2 diabetes, there are indications of genetic and epigenetic influences. This thesis has focused on the characterization of primary human adipocytes, who play a crucial role in the development of type 2 diabetes. Histones are important proteins in chromatin dynamics and may be one of the factors behind epigenetic inheritance. In paper I, we characterized histone variants and posttranslational modifications in human adipocytes. Several of the specific posttranslational histone modifications we identified have been characterized in other cell types, but the majority was not previously known. Moreover, we identified a variant of histone H4 on protein level for the first time. In paper II, we studied specific histone H3 methylations in the adipocytes. We found that overweight is correlated with a reduction of H3K4me2 while type 2 diabetes is associated with an increase of H3K4me3. This shows a genome-wide difference in important chromatin modifications that could help explain the epidemiologically shown association between epigenetics and metabolic health. Caveolae is a plasma membrane structure involved in the initial and important steps of insulin signaling. In paper III we characterized the IQGAP1 interactome in human adipocytes and suggest that IQGAP1 is a link between caveolae and the cytoskeleton. Moreover, the amount of IQGAP1 is drastically lower in adipocytes from type 2 diabetic subjects compared with controls implying a potential role for IQGAP1 in insulin resistance. In conclusion, this thesis provides new insights into the insulin signaling frameworks and the histone variants and modifications of human adipocytes

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A mass-tolerant database search identifies a large proportion of unassigned spectra in shotgun proteomics as modified peptides

Cited by 382 publications

References 43 publications

Mapping Proteoforms and Protein Complexes From King Cobra Venom Using Both Denaturing and Native Top-down Proteomics

Mapping Proteoforms and Protein Complexes From King Cobra Venom Using Both Denaturing and Native Top-down Proteomics

Exploring the potential of public proteomics data

Human Adipocytes : Proteomic Approaches

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