Peptide fractionation by acid pH SDS‐free electrophoresis

Abstract

SDS-free polyacrylamide gel electrophoresis is an effective alternative approach to peptide fractionation. Here we describe a discontinuous buffer system at acid pH that improves the separation of acidic peptides from tryptic digestion. MOPS and chloride act as trailing and leading ions, respectively, in this system, while histidine operates as counterion and buffers all solutions. In these electrophoretic conditions, peptides with pI below 5.5 migrate with low overall electrophoretic mobilities but high differences from one another, which allows for their efficient resolution. In silico analysis of several proteomes shows that the acid pH system allows a peptide simplification of 2.5-fold with respect to the total peptide mixture, and still a proteome coverage of about 95% is achievable. A straightforward method with a protocol including proteomic studies was achieved for SDS-PAGE of proteins, enzyme treatment and further peptide fractionation by SDS-free acid PAGE.

Comparative proteomics analysis of the antitumor effect of CIGB-552 peptide in HT-29 colon adenocarcinoma cells

“…The discontinuous buffer system A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 7 MOPS/histidine described previously [11,15] was used. For this electrophoresis, the gels were polymerized on a tube that is vertically inserted between two compartments containing the cathode and anode buffers.…”

“…Similar patterns of protein bands were obtained after the SDS-PAGE analysis of nuclear fractions of both, control and CIGB-552 treated cells (See Figure 3 in [13]). The study was performed using the DF-PAGE method [10,11,15] (See Figure 2 in [13]). DF-PAGE combines protein fractionation by SDS-PAGE, ingel protein digestion and peptide fractionation by SDS-free PAGE.…”

A scoring model for phosphopeptide site localization and its impact on the question of whether to use MSA

“…In this regard, there are a number of existing methods for enriching for phosphorylated peptides; for an in-depth understanding of the methods related to phosphoproteomics we recommend reviews that address widely adopted strategies, such as Immobilized Metal Affinity Chromatography, Titanium Dioxide [25][26][27], and even more general fractionation strategies [28]. In this work, we relied on the hydroxyapatite (HAP) chromatography for enriching for phosphopeptides.…”

In-depth analysis of protein inference algorithms using multiple search engines and well-defined metrics

“…The field has seen great development in the last years due to advances in mass spectrometry (MS) instrumentation, the development of new analytical methods (3)(4)(5), and novel computational approaches (2,6). Bottom-up proteomics is currently the standard analytical method to identify and quantify proteins based on the presence of peptides obtained by digestion of the protein mix during sample preparation.…”