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Green bars above refer to peptide coverage.
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This is a case where oxidative modification of Met residues, which are often located in transmembrane segments, leads to increased peptide coverage, which is often an issue when analyzing membrane proteins by LC-MS.
Two proteases, pepsin and fungal XIII, were used for protein digestion to achieve high peptide coverage.
SEQUEST software (Thermo Finnigan) combined with DXMS Explorer (Sierra Analytics) were used to generate the peptide coverage maps for different quench conditions, and the best one was used for the H/H exchange experiment.
Analysis for PTM signatures in MSMS spectra for all identified proteins did not reveal any clear phosphorylation presence in neither STEM profile proteins (Table 2) or between groups differentially expressed proteins (Table 1), possibly due to lack of whole peptide coverage after trypsin digestion.
We considered identified proteins with greater than 10% peptide coverage to be a synaptic candidate ubiquitinated protein (synCUP) if they also had a Tg/non-Tg spec ratio greater than 2. A total of 279 proteins met these criteria (Table S2 and S3).
To compare the mucin peptide coverage, we have only considered the nonglycosylated regions of the protein.
The frequent observation of multiple proteins in single gel features was attributed to the sensitivity and greater peptide coverage that can be achieved with nano-LC-MS methods as compared to, for example, MALDI-MS analysis of peptides from gel features.
Thus, it is important to consider protein size and peptide coverage when assessing proteins with relatively low peptide scores (e.g. two peptides against zero background).
Total peptide coverage corresponded to 47% of the predicted peptide sequence with a ProFound expectation score of 2.5 × 10-6 [ 11].
Normally, extensive fractionation strategies or relative enrichment methods are used to improve the proteome and peptide coverage.
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