title: Pros and cons of peptide isolectric focusing in shotgun proteomics.
abstract: In shotgun proteomics, protein mixtures are proteolytically digested before tandem mass spectrometry (MS/MS) analysis. Biological samples are generally characterized by a very high complexity, therefore a step of peptides fractionation before the MS analysis is essential. This passage reduces the sample complexity and increases its compatibility with the sampling performance of the instrument. Among all the existing approaches for peptide fractionation, isoelectric focusing has several peculiarities that are theoretically known but practically rarely exploited by the proteomics community. The main aim of this review is to draw the readers' attention to these unique qualities, which are not accessible with other common approaches, and that represent important tools to increase confidence in the identification of proteins and some post-translational modifications. The general characteristics of different methods to perform peptide isoelectric focusing with natural and artificial pH gradients, the existing instrumentation, and the informatics tools available for isoelectric point calculation are also critically described. Finally, we give some general conclusions on this strategy, underlying its principal limitations.
authors: Department of Medicine, University of Padova, Via Giustiniani 2, 35121 Padova, Italy; Proteomics Center of Padova University, VIMM and Padova University Hospital, Via G. Orus 2/B, 35129 Padova, Italy. millionirenato@gmail.com
authors: Renato
authors: R
authors: Millioni
authors: Cinzia
authors: C
authors: Franchin
authors: Paolo
authors: P
authors: Tessari
authors: Rita
authors: R
authors: Polati
authors: Daniela
authors: D
authors: Cecconi
authors: Giorgio
authors: G
authors: Arrigoni
mesh_terms: D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods*; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000032:analysis* / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*
chemical_list: D010455:Peptides