Proteomics Based on Peptide Fractionation by SDS-Free PAGE

Abstract

Here we demonstrate the usefulness of peptide fractionation by SDS-free polyacrylamide gel electrophoresis and its applicability to proteomics studies. In the absence of SDS, the driving force for the electrophoretic migration toward the anode is supplied by negatively charged acidic amino acid residues and other residues as phosphate, sulfate and sialic acid, while the resulting mobility depends on both the charge and the molecular mass of the peptides. A straightforward method was achieved for SDS-PAGE of proteins, enzyme digestion, peptide transfer and fractionation by SDS-free PAGE, which was named dual-fractionation polyacrylamide gel electrophoresis (DF-PAGE). This method increases the number of identified proteins 2.5-fold with respect to the proteins identified after direct analysis, and more than 80% of assigned peptides were found in unique SDS-free gel slices. A vast majority of identified peptides (93%) have p I values below 7.0, and 7% have p I values between 7.0 and 7.35. Peptide digests that were derived from complex protein mixtures were in consequence simplified as peptides that are positively charged are not recovered in the present conditions. The analysis of a membrane protein extract from Neisseria meningitidis by this approach allowed the identification of 97 proteins, including low-abundance components.

Peptide fractionation by acid pH SDS‐free electrophoresis

“…In model protein mixtures as a, b, and k-casein as well as egg white, which contain very acidic tryptic peptides including phosphopeptides, several signals were identified in the bands next to bromophenol blue front (Supporting Information I). In agreement with the results at basic pH [18], very acidic peptides and phosphopeptides were detected in the slice containing the fastest migrating species. Interestingly, using the new discontinuous buffer system, fewer nonphosphorylated peptides were detected in this fraction for both samples (caseins and egg white mixture) and the signalto-noise ratio in the spectrum was also improved.…”

“…Recently, we reported the use of SDS-free PAGE for peptide fractionation and its usefulness for proteomic studies [18] in combination with a first dimension for protein separation via SDS-PAGE. Peptides fractionated by SDS-free PAGE on the Ornstein gel system had theoretical isoelectric points ranging between 3.0 and 7.3 [18], which suggested that manipulating the pH of the buffer should allow fractionating a different subset of peptides. In the present work, we evaluated the effect of pH on the migration of peptides and developed a discontinuous buffer system to select and simultaneously fractionate peptides with pI lower than 5.5.…”

“…Larger format gels have also been successfully used. Figure 1 compares the electrophoretic separation of peptides derived from a tryptic digest of streptokinase (47 kDa) at basic [18] versus acid pH. In this experiment, lanes were cut into eight slices; peptides from each slice were eluted independently and analyzed by ESI-MS.…”

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.…”

Design of a software for calculating isoelectric point of a polypeptide according to their net charge using the graphical programming language LabVIEW

“…The existing comparison has been meager, using limited data [17], or peptides instead of proteins [18,19] or inaccurately reports [17,20]. In other words the pI is obtained incidentally by isoelectric focusing assays, free flow electrophoresis, capillary electrophoresis and gel electrophoresis [21][22][23].…”