H-Score, a Mass Accuracy Driven Rescoring Approach for Improved Peptide Identification in Modification Rich Samples

Savitski, Mikhail M.; Mathieson, Toby; Becher, Isabelle; Bantscheff, Marcus

doi:10.1021/pr1006813

Cited by 33 publications

(44 citation statements)

References 21 publications

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“…Fragmentation spectra of highly charged and high M w precursors potentially contain multiply charged fragment ions, which are often weighted differently/weaker than singly charged ions by certain search engines. 63 We applied the H-Score script to our data, 64 searched with the digestion specificity that was dictated by our observation (summarized above, in Figure 5 for Asp-N and similarly in Figure S-9 for the other digestions). We compared the number of unique peptides identified, XCorr distribution, and M w median for increasing M w ranges (Figure 5 for Asp-N and Figure S-9 for all data, Table S-6).…”

Section: Resultsmentioning

confidence: 99%

Toward an Optimized Workflow for Middle-Down Proteomics

et al. 2017

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Mass spectrometry (MS)-based proteomics workflows can crudely be classified into two distinct regimes, targeting either relatively small peptides (i.e., 0.7 kDa < Mw < 3.0 kDa) or small to medium sized intact proteins (i.e., 10 kDa < Mw < 30 kDa), respectively, termed bottom-up and top-down proteomics. Recently, a niche has started to be explored covering the analysis of middle-range peptides (i.e., 3.0 kDa < Mw < 10 kDa), aptly termed middle-down proteomics. Although middle-down proteomics can follow, in principle, a modular workflow similar to that of bottom-up proteomics, we hypothesized that each of these modules would benefit from targeted optimization to improve its overall performance in the analysis of middle-range sized peptides. Hence, to generate middle-range sized peptides from cellular lysates, we explored the use of the proteases Asp-N and Glu-C and a nonenzymatic acid induced cleavage. To increase the depth of the proteome, a strong cation exchange (SCX) separation, carefully tuned to improve the separation of longer peptides, combined with reversed phase-liquid chromatography (RP-LC) using columns packed with material possessing a larger pore size, was used. Finally, after evaluating the combination of potentially beneficial MS settings, we also assessed the peptide fragmentation techniques, including higher-energy collision dissociation (HCD), electron-transfer dissociation (ETD), and electron-transfer combined with higher-energy collision dissociation (EThcD), for characterization of middle-range sized peptides. These combined improvements clearly improve the detection and sequence coverage of middle-range peptides and should guide researchers to explore further how middle-down proteomics may lead to an improved proteome coverage, beneficial for, among other things, the enhanced analysis of (co-occurring) post-translational modifications.

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Section: Resultsmentioning

confidence: 99%

Toward an Optimized Workflow for Middle-Down Proteomics

et al. 2017

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“…Single-fraction peak lists generated from each method were then merged into one larger peak list for database searching using inhouse scripts, in which CID peak lists was further filtered as minimum fragment ions was set to 100, maximum number of fragment ion count was set to 100, maximum number of fragment ions was set to 100. Peak list files from HCD were deisotoped and charge deconvoluted as described (52,53). Peak lists were searched against a IPI human database (version 3.52, 69,164 sequences; 29,064,824 residues) and its decoy database created by Mascot using Mascot software version 2.3.02 (Matrix Science, UK).…”

Section: Methodsmentioning

confidence: 99%

Enhancing the Identification of Phosphopeptides from Putative Basophilic Kinase Substrates Using Ti (IV) Based IMAC Enrichment

Zhou

Low

Hennrich

et al. 2011

Molecular & Cellular Proteomics

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Metal and metal oxide chelating-based phosphopeptide enrichment technologies provide powerful tools for the in-depth profiling of phosphoproteomes. One weakness inherent to current enrichment strategies is poor binding of phosphopeptides containing multiple basic residues. The problem is exacerbated when strong cation exchange (SCX) is used for pre-fractionation, as under low pH SCX conditions phosphorylated peptides with multiple basic residues elute with the bulk of the tryptic digest and therefore require more stringent enrichment. Here, we report a systematic evaluation of the characteristics of a novel phosphopeptide enrichment approach based on a combination of low pH SCX and Ti 4؉ -immobilized metal ion affinity chromatography (IMAC) comparing it one-to-one with the well established low pH SCX-TiO 2 enrichment method. We also examined the effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFP), trifluoroacetic acid (TFA), or 2,5-dihydroxybenzoic acid (DHB) in the loading buffer, as it has been hypothesized that high levels of TFA and the perfluorinated solvent HFP improves the enrichment of phosphopeptides containing multiple basic residues. We found that Ti 4؉ -IMAC in combination with TFA in the loading buffer, outperformed all other methods tested, enabling the identification of around 5000 unique phosphopeptides containing multiple basic residues from 400 g of a HeLa cell lysate digest. In comparison, ϳ2000 unique phosphopeptides could be identified by Ti 4؉ -IMAC with HFP and close to 3000 by TiO 2 . We confirmed, by motif analysis, the basic phosphopeptides enrich the number of putative basophilic kinases substrates. In addition, we performed an experiment using the SCX/Ti 4؉ -IMAC methodology alongside the use of collision-induced dissocia- Reversible protein phosphorylation widely regulates cellular functions through protein kinases and phosphatases (1, 2). Determination and a quantitative analysis of phosphorylation sites are a prerequisite for unraveling regulatory processes and signaling networks (3-6). The analytical methods of choice for characterizing protein phosphorylation have shifted from traditional methods such as radioactive labeling and gel electrophoresis to advanced mass spectrometry, a highthroughput technology (7). It has been estimated that ϳ30% of cellular proteins are phosphorylated during the life cycle of the cell (8). There has been a continuing intense focus on developing enrichment and phosphopeptide sequencing strategies to facilitate the large-scale profiling of phosphorylation events. Currently, one of the most commonly adopted strategies is the use of two sequential steps of chromatographic based separations; an initial fractionation step for reducing sample complexity and, subsequently, a more specific enrichment of phosphopeptides.Typically, low-pH strong cation exchange (SCX) 1 chromatography is used as the first step where peptides are fraction- 1 The abbreviations used are: SCX, strong cation exchange chromatography; Ti 4ϩ -IMAC, immobilized titanium (IV) ion affinity ...

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“…When both HCD and ETD scans were presents in the same raw files, separate mgfs for the two fragmentation techniques were created. All high-resolution HCD and ETD MS/MS spectra were deconvoluted using MS Spectrum Processor v0.9 24 . In the ETD spectra the precursor, the charge-reduced precursor(s) and the neutral losses were removed.…”

Section: Database Analysis and Configuration Of Mascot Modificationsmentioning

confidence: 99%

Optimization of LTQ-Orbitrap Mass Spectrometer Parameters for the Identification of ADP-Ribosylation Sites

et al. 2015

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ADP-ribosylation of proteins alters their function or provides a scaffold for the recruitment of other proteins, thereby regulating several important cellular processes. Mono- or poly-ADP-ribosylation is catalyzed by different ADP-ribosyltransferases (ARTs) that have different subcellular localizations and modify different amino acid acceptor sites. However, our knowledge of ADP-ribosylated proteins and their acceptor amino acids is still limited due to the lack of suitable mass spectrometry (MS) tools. Here, we describe an MS approach for the detection of ADP-ribosylated peptides and identification of the ADP-ribose acceptor sites, combining higher-energy collisional dissociation (HCD) and electron-transfer dissociation (ETD) on an LTQ-Orbitrap mass spectrometer. The presence of diagnostic ions of ADP-ribose in the HCD spectra allowed us to detect putative ADP-ribosylated peptides to target in a second LC-MS/MS analysis. The combination of HCD with ETD fragmentation gave a more comprehensive coverage of ADP-ribosylation sites than that with HCD alone. We successfully identified different ADP-ribose acceptor sites on several in vitro modified proteins. The combination of optimized HCD and ETD methods may be applied to complex samples, allowing comprehensive identification of ADP-ribosylation acceptor sites.

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H-Score, a Mass Accuracy Driven Rescoring Approach for Improved Peptide Identification in Modification Rich Samples

Cited by 33 publications

References 21 publications

Toward an Optimized Workflow for Middle-Down Proteomics

Toward an Optimized Workflow for Middle-Down Proteomics

Enhancing the Identification of Phosphopeptides from Putative Basophilic Kinase Substrates Using Ti (IV) Based IMAC Enrichment

Optimization of LTQ-Orbitrap Mass Spectrometer Parameters for the Identification of ADP-Ribosylation Sites

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