Label-free detection of differential protein expression by LC/MALDI mass spectrometry.
Protein abundance changes during disease or experimental perturbation are increasingly analyzed by label-free LC/MS approaches. Here we demonstrate the use of LC/MALDI MS for label-free detection of protein expression differences using Escherichia coli cultures grown on arabinose, fructose or glucose as a carbon source. The advantages of MALDI, such as detection of only singly charged ions, and MALDI plate archiving to facilitate retrospective MS/MS data collection are illustrated. MALDI spectra from RP chromatography of tryptic digests of the E. coli lysates were aligned and quantitated using the Rosetta Elucidator system. Approximately 5000 peptide signals were detected in all LC/MALDI runs spanning over 3 orders of magnitude of signal intensity. The average coefficients of variation for all signals across the entire intensity range in all technical replicates were found to be <25%. Pearson correlation coefficients from 0.93 to 0.98 for pairwise comparisons illustrate high replicate reproducibility. Expression differences determined by Analysis of Variance highlighted over 500 isotope clusters ( p < 0.01), which represented candidates for targeted peptide identification using MS/MS. Biologically interpretable protein identifications that could be derived underpin the general utility of this label-free LC/MALDI strategy.
Neubert
Hendrik
H
Pfizer Global Research and Development, Sandwich, Kent CT13 9NJ, United Kingdom. hendrik.neubert@pfizer.com
Bonnert
Timothy P
TP
Rumpel
Klaus
K
Hunt
Brandon T
BT
Henle
Ernst S
ES
James
Ian T
IT
D001089:Arabinose / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D019295:Computational Biology; D001330:Electronic Data Processing; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000032:analysis / Q000378:metabolism; D005632:Fructose / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D025341:Principal Component Analysis; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry
D029968:Escherichia coli Proteins; D011506:Proteins; D020543:Proteome; D005632:Fructose; D001089:Arabinose; D014357:Trypsin; D005947:Glucose