High-pH reversed-phase chromatography with fraction concatenation for 2D proteomic analysis

Abstract

Summary Orthogonal high-resolution separations are critical for attaining improved analytical dynamic range and protein coverage in proteomic measurements. High pH reversed-phase liquid chromatography (RPLC) followed by fraction concatenation affords better peptide analysis than conventional strong-cation exchange (SCX) chromatography applied for the two-dimensional proteomic analysis. For example, concatenated high pH reversed-phase liquid chromatography increased identification for peptides (1.8-fold) and proteins (1.6-fold) in shotgun proteomics analyses of a digested human protein sample. Additional advantages of high pH RPLC with fraction concatenation include improved protein sequence coverage, simplified sample processing, and reduced sample losses, making this an attractive alternative to SCX chromatography in conjunction with the second dimension low pH RPLC for two-dimensional proteomics analyses.

Proteomic Analysis Reveals Branch-specific Regulation of the Unfolded Protein Response by Nonsense-mediated mRNA Decay

“…Elution was performed at a flow rate of 0.1 ml/min using a gradient of mobile phase A (20 mM ammonium formate, pH 10) and B (acetonitrile), from 1% to 37.5% over 61 min. Fractions were collected every 2 min across the entire gradient length and concatenated into 10 final samples as discussed previously (33). Fractions were dried in a SpeedVac centrifuge and reconstituted in 0.1% formic acid prior to mass spectrometry (MS) analysis.…”

Refined Preparation and Use of Anti-diglycine Remnant (K-ε-GG) Antibody Enables Routine Quantification of 10,000s of Ubiquitination Sites in Single Proteomics Experiments

“…Additionally, pooling of nonadjacent basic RP fractions generates highly uniform and nonredundant samples for K--GG enrichment that enables consistent depth-of-coverage for K--GG peptides across all eight fractions (Fig. 3b) (18,19). The eight basic RP fractions are directly enriched for K--GG peptides using 31 g of DMP cross-linked anti-K--GG antibody per fraction (Fig.…”

Quantitative analysis of the murine lipid droplet-associated proteome during diet-induced hepatic steatosis

“…Peptide-containing fractions were divided into two equal numbered groups, "early" and "late". The fi rst early fraction was concatenated with the fi rst late fraction, and so on ( 18 ). Concatenated samples were dried in vacuo, resuspended in load solvent (98:2:0.01, water:acetonitrile:formic acid), and 1.5 g aliquots were run on a Velos Orbitrap mass spectrometer (Thermo Fisher Scientifi c, Inc.) as described previously, with the exception that the activation energy was 40 ms ( 19 ).…”