Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: implications for proteome mapping.
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.
Ogorzalek Loo
R R
RR
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0674, USA.
Mitchell
C
C
Stevenson
T I
TI
Martin
S A
SA
Hines
W M
WM
Juhasz
P
P
Patterson
D H
DH
Peltier
J M
JM
Loo
J A
JA
Andrews
P C
PC
D000180:Acrylic Resins; D000818:Animals; D002417:Cattle; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D005782:Gels; D010449:Peptide Mapping; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling
D000180:Acrylic Resins; D005782:Gels; D011506:Proteins; C016680:polyacrylamide gels; D012967:Sodium Dodecyl Sulfate; D003488:Cyanogen Bromide