title: A novel prefractionation method combining protein and peptide isoelectric focusing in immobilized pH gradient strips.
abstract: To increase sensitivity and analytical depth in shotgun proteomics, prefractionation of complex samples is often used. Here we describe a novel prefractionation method, Sandwich high resolution isoelectric focusing, which combines both protein and peptide isoelectric focusing. In the first step, intact proteins are separated on the basis of isoelectric point (pI) using traditional immobilized pH gradient (IPG) strips. Segments in the IPG-strip containing proteins of interest are subsequently cut out and applied to in-strip digestion, without subsequent peptide elution. In the second peptide isoelectric focusing step, the strip segments are used as loading bridges. The peptides are thereby directly applied to the peptide isoelectric focusing, without an intermediate elution step, and separated on narrow range IPG strips to reduce the complexity on the peptide level. In the final step, the peptides are eluted into 96-well plates and analyzed with mass spectrometry. In a proof of principle experiment, using this method to zoom in on pI regions of interest in human plasma, we identify over 800 proteins, with concentrations spanning over 6 orders of magnitude.
authors.affiliation: Cancer Proteomics Mass Spectrometry, Department of Oncology-Pathology, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden. maria.pernemalm@ki.se
authors.forename: Maria
authors.initials: M
authors.lastname: Pernemalm
authors.forename: Janne
authors.initials: J
authors.lastname: Lehtiö
mesh_terms: D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry; D005591:Chemical Fractionation / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D059748:Proteolysis; D040901:Proteomics; D018892:Proton-Motive Force; D011934:Reagent Strips; D014357:Trypsin / Q000737:chemistry
chemical_list: D001798:Blood Proteins; D010446:Peptide Fragments; D011934:Reagent Strips; D014357:Trypsin