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.

• Klíčová slova
• Data-independent acquisition, Isolation windows, Maxquant, Spectronaut, pFind,
• MeSH
• chromatografie s reverzní fází MeSH
• hmotnostní spektrometrie metody   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• peptidy analýza   MeSH
• proteiny analýza   MeSH
• proteomika metody   MeSH
• software MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• peptidy MeSH
• proteiny MeSH

Reliable determination of protein complex composition or changes to protein levels in whole cells is challenging. Despite the multitude of methods now available for labeling, analysis, and the statistical processing of data, this large variety is of itself an issue: Which approach is most appropriate, where do you set cutoffs, and what is the most cost-effective strategy? One size does not fit all for such work, but some guidelines can help in terms of reducing cost, improving data quality, and ultimately advancing investigations. Here we describe two protocols and algorithms for facile sample preparation for mass spectrometric analysis, robust data processing, and considerations of how to interpret large proteomic datasets in a productive and robust manner.

• Klíčová slova
• Data presentation, MaxQuant, Proteomics, SDS-PAGE, Sample preparation, Trypanosoma,
• MeSH
• datové soubory jako téma MeSH
• elektroforéza v polyakrylamidovém gelu metody   MeSH
• hmotnostní spektrometrie metody   MeSH
• multiproteinové komplexy izolace a purifikace   metabolismus   MeSH
• proteomika metody   MeSH
• protozoální proteiny izolace a purifikace   metabolismus   MeSH
• Trypanosoma brucei brucei metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• multiproteinové komplexy MeSH
• protozoální proteiny MeSH

Phosphorylation is among the most important post-translational modifications of proteins and has numerous regulatory functions across all domains of life. However, phosphorylation is often substoichiometric, requiring selective and sensitive methods to enrich phosphorylated peptides from complex cellular digests. Various methods have been devised for this purpose and we have recently described a Fe-IMAC HPLC column chromatography setup which is capable of comprehensive, reproducible, and selective enrichment of phosphopeptides out of complex peptide mixtures. In contrast to other formats such as StageTips or batch incubations using TiO2 or Ti-IMAC beads, Fe-IMAC HPLC columns do not suffer from issues regarding incomplete phosphopeptide binding or elution and enrichment efficiency scales linearly with the amount of starting material. Here, we provide a step-by-step protocol for the entire phosphopeptide enrichment procedure including sample preparation (lysis, digestion, desalting), Fe-IMAC column chromatography (column setup, operation, charging), measurement by LC-MS/MS (nHPLC gradient, MS parameters) and data analysis (MaxQuant). To increase throughput, we have optimized several key steps such as the gradient time of the Fe-IMAC separation (15 min per enrichment), the number of consecutive enrichments possible between two chargings (>20) and the column recharging itself (<1 h). We show that the application of this protocol enables the selective (>90 %) identification of more than 10,000 unique phosphopeptides from 1 mg of HeLa digest within 2 h of measurement time (Q Exactive Plus).

• Klíčová slova
• LC-MS, Phosphocapture, Phosphorylation, Proteomics,
• MeSH
• buněčné linie MeSH
• chromatografie kapalinová MeSH
• fosfopeptidy MeSH
• fosfoproteiny * MeSH
• imidazoly chemie   MeSH
• lidé MeSH
• proteom * MeSH
• proteomika metody   MeSH
• průběh práce MeSH
• software MeSH
• statistika jako téma MeSH
• tandemová hmotnostní spektrometrie MeSH
• železo chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fosfopeptidy MeSH
• fosfoproteiny * MeSH
• imidazoleacetic acid MeSH Prohlížeč
• imidazoly MeSH
• proteom * MeSH
• železo MeSH