Optimization of MALDI-TOF MS detection for enhanced sensitivity of affinity-captured proteins spanning a 100 kDa mass range.
Analysis of complex biological samples by MALDI-TOF mass spectrometry has been generally limited to the detection of low-mass protein (or protein fragment) peaks. We have extended the mass range of MALDI-TOF high-sensitivity detection by an order of magnitude through the combined optimization of instrument parameters, data processing, and sample preparation procedures for affinity capture. WCX, C3, and IMAC magnetic beads were determined to be complementary and most favorable for broad mass range protein profiling. Key instrument parameters for extending mass range included adjustment of the ADC offset and preamplifier filter values of the TOF detector. Data processing was improved by a combination of constant and quadratic down-sampling, preceded by exponential baseline subtraction, to increase sensitivity of signal peaks. This enhancement in broad mass range detection of protein signals will be of direct benefit in MS expression profiling studies requiring full linear range mass detection.
Gatlin-Bunai
Christine L
CL
Department of Applied Science, the College of William and Mary, Williamsburg, Virginia 23185, USA. clbuna@wm.edu
Cazares
Lisa H
LH
Cooke
William E
WE
Semmes
Oliver J
OJ
Malyarenko
Dariya I
DI
D001798:Blood Proteins / Q000737:chemistry; D002138:Calibration; D006801:Humans; D008280:Magnetics; D013058:Mass Spectrometry; D008970:Molecular Weight; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D040081:Protein Array Analysis; D011506:Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D001798:Blood Proteins; D010455:Peptides; D011506:Proteins