Value of Using Multiple Proteases for Large-Scale Mass Spectrometry-Based Proteomics

Swaney, Danielle L.; Wenger, Craig D.; Coon, Joshua J.

doi:10.1021/pr900863u

Cited by 422 publications

(492 citation statements)

References 32 publications

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“…For Drosophila melanogaster, adding a fractionated adult sample to the spectral library increased the number of proteins in the library from 5,335 to 6,040 showing that adding specific tissue/developmental stage is useful to increase the size of spectral libraries for SWATH-MS. The peptide size distributions are very similar for both spectral libraries with a high proportion of peptides around 10 amino acids long ( Figure S2) which is in agreement with previous study using trypsin in yeast [23]. Our spectral libraries cover around 34% and 15% of the expected proteomes of Drosophila melanogaster and Solanum lycopersicum which, to our knowledge, constitute the most complete spectral libraries acquired on TripleTOF instruments for either species.…”

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confidence: 89%

Spectral Libraries for SWATH‐MS Assays for Drosophila melanogaster and Solanum lycopersicum

et al. 2017

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Quantitative proteomics methods have emerged as powerful tools for measuring protein expression changes at the proteome level. Using mass-spectrometry (MS) based approaches, it is now possible to routinely quantify thousands of proteins. However, pre-fractionation of the samples at the protein or peptide level is usually necessary to go deep into the proteome, increasing both MS analysis time and technical variability. Recently, a new MS acquisition method named SWATH was introduced with the potential to provide good coverage of the proteome as well as a good measurement precision without prior sample fractionation. In contrast to shotgun based MS however, a library containing experimental acquired spectra is necessary for the bioinformatics analysis of SWATH data. In this study, we built spectral libraries for two widely used models to study crop ripening or animal embryogenesis, Solanum lycopersicum (tomato) and Drosophila melanogaster, respectively. The spectral libraries comprise fragments for 5,197 and 6,040 proteins for Solanum lycopersicum and Drosophila melanogaster, respectively, and allow reproducible quantification for thousands of peptides per MS analysis. The spectral libraries and all massspectrometry data are available in the MassIVE repository with the dataset identifiers MSV000081074 and MSV000081075 and the PRIDE repository with the dataset identifiers PXD006493 and PXD006495. For DIA, the spectral library contains experimental acquired spectra of the precursors and can be produced directly from the SWATH data, by reconstituting the MSMS spectra using the retention time of the precursors and the fragments [7], or by DDA analysis of the sample, which can be fractionated to increase the number of spectra in the library [9]. Ideally, the spectral library should be generated on a MS instrument similar to the one used to acquire further SWATH-MS data as the correlation of the fragment intensities for a peptide acquired on different instrument was shown to be rather low [10]. Although Rosenberger et al. recently published a library containing assays for 10,000 human proteins [9] and deep libraries for other applications such as SRM (Selected Reaction Monitoring) have been generated [11][12][13], the number of publicly available spectral libraries for SWATH-MS is still limited. Several other studies have produced libraries for other species [14][15][16]. However, spectral libraries are still missing for many species and, when available, the size of the libraries are limited and a deeper coverage of the proteome would increase the number of potential identifications in SWATH-MS analysis. In the present study, we produced spectral libraries for two well established models to study fruit ripening or animal embryogenesis, respectively Solanum lycopersicum L. and Drosophila melanogaster, for which no or low depth spectral libraries for SWATH-MS on TripleTOF instruments have been produced so far [17,18]. These libraries contain assays for more than 5,000 proteins for both species and are best suited...

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confidence: 89%

Spectral Libraries for SWATH‐MS Assays for Drosophila melanogaster and Solanum lycopersicum

et al. 2017

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“…Alkylation was quenched by a 15-min incubation in 5 mM dithiothreitol at room temperature. This was followed by a multiple protease approach, utilized to maximize protein sequence coverage (45), and involved addition of five different proteases (individually or sequentially) (see the details in the supplemental Experimental Procedures).…”

Section: Digestion Of Pro-␣1(v) and ␣1(v) Samples For Mass Spectrometmentioning

confidence: 99%

“…The enabling technology was an ETD-enabled LTQ Orbitrap Velos hybrid mass spectrometer exhibiting low ppm mass accuracy, high resolution MS, and MS n capabilities combined with multiple options for performing peptide dissociation (47). We began by using multiple proteases to generate a diverse pool of peptides amenable to MS n (45). The combination of mass accuracy and a capability to perform multistage activation using three different dissociation techniques often allowed us to pinpoint the exact residue(s) carrying individual PTMs.…”

Section: A High Mass Accuracy High Resolution Tandem Mass Spectrometmentioning

confidence: 99%

Comprehensive Mass Spectrometric Mapping of the Hydroxylated Amino Acid residues of the α1(V) Collagen Chain

Yang

Park

Davis

et al. 2012

Journal of Biological Chemistry

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Background: ␣1(V) is an extensively modified collagen chain important in disease. Results: Comprehensive mapping of ␣1(V) post-translational modifications reveals unexpectedly large numbers of X-position hydroxyprolines in Gly-X-Y amino acid triplets. Conclusion:The unexpected abundance of X-position hydroxyprolines suggests a mechanism for differential modification of collagen properties. Significance: Positions, numbers, and occupancy of modified sites can provide insights into ␣1(V) biological properties.

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“…It had been shown that the use of multiple proteases could remarkly increase the sequence coverage of identified proteins for large-scale proteomics analysis. 15 The lowspecificity proteases such as elastase, proteinase K, and thermolysin had been used for in-depth mapping of phosphorylation sites of murine circadian protein period 2; a total of 21 phosphorylation sites were identified from this protein, whereas only 6 sites were identified by trypsin. 16 The low-specificity protease elastase had been used together with trypsin for phosphoproteome analysis of mitotic spindle proteins; 17 the overlap of phosphorylation sites identified by the two proteases was less than 10%.…”

Section: ■ Introductionmentioning

confidence: 99%

Improve the Coverage for the Analysis of Phosphoproteome of HeLa Cells by a Tandem Digestion Approach

Bian

Song

et al. 2012

J. Proteome Res.

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Complete coverage of all phosphorylation sites in a proteome is the ultimate goal for large-scale phosphoproteome analysis. However, only making use of one protease trypsin for protein digestion cannot cover all phosphorylation sites, because not all tryptic phosphopeptides are detectable in MS. To further increase the phosphoproteomics coverage of HeLa cells, we proposed a tandem digestion approach by using two different proteases. By combining the data set of the first Glu-C digestion and the second trypsin digestion, the tandem digestion approach resulted in the identification of 8062 unique phosphopeptides and 8507 phosphorylation sites in HeLa cells. The conventional trypsin digestion approach resulted in the identification of 3891 unique phosphopeptides and 4647 phosphorylation sites. It was found that the phosphorylation sites identified from the above two approaches were highly complementary. By combining above two data sets, in total we identified 10899 unique phosphopeptides and 11262 phosphorylation sites, corresponding to 3437 unique phosphoproteins with FDR < 1% at peptide level. We also compared the kinase motifs extracted from trypsin, Glu-C, or a second trypsin digestion data sets. It was observed that basophilic motifs were more frequently found in the trypsin and the second trypsin digestion data sets, and the acidic motifs were more frequently found in the Glu-C digestion data set. These results demonstrated that our tandem digestion approach is a good complement to the conventional trypsin digestion approach for improving the phosphoproteomics analysis coverage of HeLa cells.

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Value of Using Multiple Proteases for Large-Scale Mass Spectrometry-Based Proteomics

Cited by 422 publications

References 32 publications

Spectral Libraries for SWATH‐MS Assays for Drosophila melanogaster and Solanum lycopersicum

Spectral Libraries for SWATH‐MS Assays for Drosophila melanogaster and Solanum lycopersicum

Comprehensive Mass Spectrometric Mapping of the Hydroxylated Amino Acid residues of the α1(V) Collagen Chain

Improve the Coverage for the Analysis of Phosphoproteome of HeLa Cells by a Tandem Digestion Approach

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