Influence of the protein staining in the fast ultrasonic sample treatment for protein identification through peptide mass fingerprint and matrix-assisted laser desorption ionization time of flight mass spectrometry.
The influence of the protein staining used to visualize protein bands, after in-gel protein separation, for the correct identification of proteins by peptide mass fingerprint (PMF) after application of the ultrasonic in-gel protein protocol was studied. Coomassie brilliant blue and silver nitrate, both visible stains, and the fluorescent dyes Sypro Red and Sypro Orange were evaluated. Results obtained after comparison with the overnight in-gel protocol showed that good results, in terms of protein sequence coverage and number of peptides matched, can be obtained with anyone of the four stains studied. Two minutes of enzymatic digestion time was enough for proteins stained with coomassie blue, while 4 min was necessary when silver or Sypro stainings were employed in order to reach equivalent results to those obtained for the overnigh in-gel protein protocol. For the silver nitrate stain, the concentration of silver present in the staining solution must be 0.09% (w/v) to minimize background in the MALDI mass spectra.
Galesio
M
M
REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnología, Universidade Nova de Lisboa, Monte de Caparica, Portugal.
Vieira
D V
DV
Rial-Otero
R
R
Lodeiro
C
C
Moura
I
I
Capelo
J L
JL
D000818:Animals; D002417:Cattle; D004396:Coloring Agents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry / Q000379:methods*; D010449:Peptide Mapping; D011506:Proteins / Q000032:analysis / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013194:Staining and Labeling; D014465:Ultrasonics
D004396:Coloring Agents; D011506:Proteins; D012710:Serum Albumin, Bovine