An optimized protein in-gel digest method for reliable proteome characterization by MALDI-TOF-MS analysis.
Human lung epithelial cells (A549) were used as a model to develop a reliable proteome characterization method by peptide mass fingerprinting (PMF). Lung cell lysate proteins and protein standards were separated by 2D-gel electrophoresis, stained with Coomassie blue, gel plugs were subjected to commonly adapted as well as optimized in-gel digestion/sample preparation methods. Samples were analyzed by MALDI-TOF-MS. Optimization parameters included, use of NH(4)OAc in destaining and in-gel digestion buffers, detergent/salt removal prior to in-gel digestion, use of solvents of varying polarities (0%, 30%, 60% ACN containing 0.1% TFA) to improve peptide recoveries, matrix composition (alpha-cyano-4-hydroxycinamic acid-organic solvent combinations) and on-target salt removal. This led to enhanced mass spectral information and a sensitivity gain in the order of 6-10 fold compared to that of common procedures, yielding reliable, unambiguous protein identification with femtomol protein sensitivity by Autoflex MALDI-TOF-MS. Triplicate analyses by two analysts revealed consistent, wide range m/z values including in < 1200Da region by relieving matrix-exerted signal suppression, requiring one trial to obtain a unique protein identification with superior PMF results for the optimized method. Analyses of ten A549 proteins in replicates using the optimized method yielded fast, reliable characterization, suggesting the potential application of this method in high-throughput protein identification by PMF.
Kumarathasan
P
P
Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ont., Canada K1A 0L2.
Mohottalage
S
S
Goegan
P
P
Vincent
R
R
D002460:Cell Line; D006801:Humans; D010449:Peptide Mapping / Q000379:methods*; D020543:Proteome / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D020543:Proteome