title: Practical considerations for improving the productivity of mass spectrometry-based proteomics.
abstract: Mass spectrometry (MS) is currently the most sensitive and selective analytical technique for routine peptide and protein structure analysis. Top-down proteomics is based on tandem mass spectrometry (MS/ MS) of intact proteins, where multiply charged precursor ions are fragmented in the gas phase, typically by electron transfer or electron capture dissociation, to yield sequence-specific fragment ions. This approach is primarily used for the study of protein isoforms, including localization of post-translational modifications and identification of splice variants. Bottom-up proteomics is utilized for routine high-throughput protein identification and quantitation from complex biological samples. The proteins are first enzymatically digested into small (usually less than ca. 3 kDa) peptides, these are identified by MS or MS/MS, usually employing collisional activation techniques. To overcome the limitations of these approaches while combining their benefits, middle-down proteomics has recently emerged. Here, the proteins are digested into long (3-15 kDa) peptides via restricted proteolysis followed by the MS/MS analysis of the obtained digest. With advancements of high-resolution MS and allied techniques, routine implementation of the middle-down approach has been made possible. Herein, we present the liquid chromatography (LC)-MS/MS-based experimental design of our middle-down proteomic workflow coupled with post-LC supercharging.
authors: Biomolecular Mass Spectrometry Laboratory, Ecole Polytechnique Fédérale de Lausanne, EPFL LSMB BCH 4307, CH-1015 Lausanne.
authors: Unige A
authors: UA
authors: Laskay
authors: Kristina
authors: K
authors: Srzentić
authors: Luca
authors: L
authors: Fornelli
authors: Oxana
authors: O
authors: Upir
authors: Anton N
authors: AN
authors: Kozhinov
authors: Michel
authors: M
authors: Monod
authors: Yury O
authors: YO
authors: Tsybin
mesh_terms: D016282:Aspartic Acid Endopeptidases / Q000032:analysis*; D001426:Bacterial Proteins / Q000378:metabolism*; D002176:Candida albicans / Q000201:enzymology*; D002853:Chromatography, Liquid / Q000379:methods*; D005656:Fungal Proteins / Q000032:analysis*; D010446:Peptide Fragments / Q000032:analysis*; D040901:Proteomics; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D005656:Fungal Proteins; D010446:Peptide Fragments; D016282:Aspartic Acid Endopeptidases; C116371:Sap9 protein, Candida albicans