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Due to the technical advances of mass spectrometers, particularly increased scanning speed and higher MS/MS resolution, the use of data-independent acquisition mass spectrometry (DIA-MS) became more popular, which enables high reproducibility in both proteomic identification and quantification. The current DIA-MS methods normally cover a wide mass range, with the aim to target and identify as many peptides and proteins as possible and therefore frequently generate MS/MS spectra of high complexity. In this report, we assessed the performance and benefits of using small windows with, e.g., 5-m/z width across the peptide elution time. We further devised a new DIA method named RTwinDIA that schedules the small isolation windows in different retention time blocks, taking advantage of the fact that larger peptides are normally eluting later in reversed phase chromatography. We assessed the direct proteomic identification by using shotgun database searching tools such as MaxQuant and pFind, and also Spectronaut with an external comprehensive spectral library of human proteins. We conclude that algorithms like pFind have potential in directly analyzing DIA data acquired with small windows, and that the instrumental time and DIA cycle time, if prioritized to be spent on small windows rather than on covering a broad mass range by large windows, will improve the direct proteome coverage for new biological samples and increase the quantitative precision. These results further provide perspectives for the future convergence between DDA and DIA on faster MS analyzers.

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All the shotgun and DIA raw data was directly analyzed by MaxQuant [15] and searched against the human canonical UniProtKB/Swiss-Prot database (downloaded February 2018, 20,258 entries). Oxidation at methionine was set as variable modification, whereas carbamidomethylation at cysteine was set as a fixed modification. Up to two missed cleavages were allowed. The mixed A2780LH sample was searched following standard SILAC setting. Other parameters are kept as default in MaxQuant. Both peptide and protein level were controlled at 1% FDR [16]. The match between run function was disabled and the second peptide search function was enabled (as default).

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