Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: Implications for proteome mapping

Abstract

Matrix-assisted laser desorption ionization (MALDI) mass spectra have been obtained directly from thin-layer isoelectric focusing (IEF) gels with as little as 700 femtomoles of alpha- and beta-chain bovine hemoglobin and bovine carbonic anhydrase, and 2 picomoles of bovine trypsinogen, soybean trypsin inhibitor, and bovine serum albumin all loaded onto a single lane. By soaking the gel in a matrix solution, matrix was deposited over the entire gel surface, allowing MALDI scanning down complete lanes of the one-dimensional gel. As long as matrix crystals were deposited finely on the surface of the gel, time-lag focusing techniques were capable of ameliorating some of the mass accuracy limitations inherent in desorbing from uneven insulator surfaces with external calibration. Eleven measurements on the 5 kDa alpha-subunit proteins of lentil lectin measured over the course of 1 h and referenced to a single calibration yielded a standard deviation of 0.025%. Colloidal gold staining was found to be compatible with desorption directly from IEF and sodium dodecyl sulfate (SDS)-polyacrylamide gels. This direct approach simplifies the interface between gel electrophoresis and mass spectrometry dramatically, making the process more amenable to automation.

Top-down, bottom-up, and side-to-side proteomics with virtual 2-D gels

“…These capabilities should facilitate studies of prokaryotic glycosylation, an established modification in both bacteria and archaea, primarily to cell surface-layer proteins. [34,35] Previous studies mass analyzing intact proteins directly from dried immobilized pH gradient gels [3,5,8], searched databases employing a mass accuracy of ± 0.1-0.2% for proteins below 50 kDa in size. Generally, 0.1% accuracy or better is achievable for reasonably abundant proteins below 20 kDa in size.…”

“…(time-lag focusing) conditions [3,6], established that surface charging of the gel (an insulator) is not the major factor reducing mass accuracy when time-lag focusing is employed. Rather, mass accuracy on linear time-of-flight instruments is governed by the evenness of the surface from which ions are desorbed.…”

“…Virtual 2-D Gel Electrophoresis [2][3][4][5][6][7][8] was developed to mass measure intact, polyacrylamide gel-isolated proteins for proteomic applications. In the virtual 2-D gel method, MS is performed directly from dried isoelectric focusing (IEF) gels, substituting MALDI-MS for the SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) 2nd dimension of classical 2-D PAGE.…”

Virtual two‐dimensional gel electrophoresis of high‐density lipoproteins

“…In our previous studies mass analyzing proteins directly from dried immobilized pH gradient gels [11,13], we searched databases assuming a mass accuracy of ± 0.1-0.2% for proteins below 50 kDa in size. Generally, 0.1% accuracy or better was achievable for reasonably abundant proteins below 20 kDa in size.…”

“…These latter effects challenge current proteomic technologies. [10][11][12][13][14][15], based on combining a first dimension isoelectric focusing (IEF) separation on polyacrylamide gels with matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) surface scanning of the dried gel ( Fig. 1), was developed to address the challenges of post-translational modifications.…”