On-probe sample pretreatment for the detection of proteins above 15 KDa from whole cell bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
A rapid methodology is described for the enhancement of the signal-to-base-line (S/B) ratio of high molecular weight protein signals from whole cell bacteria analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The procedure involves depositing growing bacteria colonies from culture dishes directly onto the MALDI probe followed by treatment of the sample spot with a 2 microL aliquot of 40% ethanol prior to the addition of a ferulic acid matrix solution (12.5 mg dissolved in 17% formic acid/33% acetonitrile/50% H(2)O). Protein signals of more than 20 kDa were routinely produced from both Gram positive and Gram negative bacteria prepared in this manner. Moreover, a substantial number of intense protein signals were also produced in the more 'conventional' fingerprint region extending from 4 to 20 kDa. This approach is rapid, easy to implement into existing methodologies, and does not require any special hardware.
Madonna
A J
AJ
Department of Chemistry, Colorado School of Mines, Golden, CO 80401, USA.
Basile
F
F
Ferrer
I
I
Meetani
M A
MA
Rees
J C
JC
Voorhees
K J
KJ
D001426:Bacterial Proteins / Q000032:analysis* / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000378:metabolism* / Q000648:ultrastructure; D006094:Gram-Positive Bacteria / Q000378:metabolism* / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D010446:Peptide Fragments / Q000032:analysis*; D010449:Peptide Mapping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D001426:Bacterial Proteins; D010446:Peptide Fragments