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Protein Identification Using Nano-HPLC-MS: ESI-MS and MALDI-MS Interfaces

2011; Springer Science+Business Media; Linguagem: Inglês

10.1007/978-1-61779-319-6_3

1940-6029

Rui Vitorino, Jana Křenková, František Foret, Pedro Domíngues, Francisco Amado,

Metabolomics and Mass Spectrometry Studies

Body fluids and body tissues have a myriad of peptides and proteins that, very often, the traditional methodologies of proteomics, such as conventional gel electrophoresis or mass spectrometry, are unable to characterize. We describe two protocols to characterize high molecular weight peptides (>3 kDa) and intact proteins involving on-line trypsin digestion, separation of the digests by nano-HPLC, and analysis by mass spectrometry using two different ionization sources (matrix-assisted laser desorption and electrospray ionization). These protocols have the advantage of promoting protein denaturation in an aqueous-organic solvent, which reduces the derivatization of the sample and facilitates an in-depth analysis for detection and identification of proteins. Additional advantages include the following: (1) integration of these protocols into standard proteomic workflows after the preprocessing of samples and separation; (2) use of high-resolution monolithic columns; and (3) the ability to acquire information from minimal amounts of sample.