Mass spectrometry‐based pseudo‐western blots

Abstract

Listen

Translate article

Modern systems biology utilizes protein mass spectrometry (MS) to quantify thousands of proteins in a given tissue. The alternative to MS, western blotting, is low‐throughput, but gives quantitative information as a function of protein molecular weight, providing critical information about protein modifications. Here, we describe an approach that combines the advantages of both: “pseudo‐western blots” (pWBs). Protein extracts are separated by standard SDS‐PAGE, and each lane is sliced into 40 successive blocks followed by in‐gel trypsin digestion. Individual samples are analyzed by label‐free quantitative LC‐MS/MS (Velos‐Orbitrap). The resulting data were then translated into a western blot‐like format (abundance vs. slice number) using a heat map generator. To establish proof‐of‐principle, we analyzed kidney inner medullas from rats subjected to high‐ or low‐water intake for 3 days. The data were assembled into hundreds of pWBs. pWBs of the vasopressin‐regulated water channel aquaporin‐2 (Aqp2) and a urea channel (Slc14a2) matched corresponding antibody‐based western blots and reproduced changes. Our results show the feasibility of the pWB approach for systems biology applications. (G.L. supported by NIBIB Biomedical Engineering Student Internship Program, 2012)