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

✍ Scribed by Rachel R. Ogorzalek Loo; Charles Mitchell; Tracy I. Stevenson; Stephen A. Martin; Wade M. Hines; Peter Juhasz; Dale H. Patterson; John M. Peltier; Joseph A. Loo; Dr. Philip C. Andrews

• Book ID: 102833468
• Publisher: John Wiley and Sons
• Year: 1997
• Tongue: English
• Weight: 977 KB
• Volume: 18
• Category: Article
• ISSN: 0173-0835
• DOI: 10.1002/elps.1150180312

✦ Synopsis

Matrix-assisted laser desorption ionization (MALDI) mass spectra have beeri obtained directly from thin-layer isoelectric focusing (IEF) gels with as little as 700 femtomoles of a-and fi-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 a-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.