Characterizing the reproducibility of a protein profiling method for the analysis of mouse bronchoalveolar lavage fluid.
The detection of biomarkers in biological fluids has been advanced by the introduction of mass spectrometry screening methods such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), which enables the detection of the presence and the molecular mass of proteins in unfractionated mixtures. The generation of reproducible mass spectra over the course of an experiment is vital in obtaining data in which differences in protein profiles between diseased and healthy states can be assessed correctly. We have developed a protocol to automate the collection of protein profiling data from a large number of samples using MALDI-TOFMS, and we used these samples to characterize the technical reproducibility of the method. This protocol has been used for the analysis of proteins found in bronchoalveolar lavage fluid samples from mice with the ultimate goal of enabling the discovery of differential expression patterns predictive of the development of chronic obstructive pulmonary disease. Samples were purified using magnetic bead-based technology and analyzed on an AnchorChip target plate. Our results demonstrate that the number of peaks detected reproducibly decreases significantly as sample size increases, which motivates the need for technical replicates to be explicitly included in the analysis of MALDI-TOF-based protein profiling studies.
McLachlan
Anne
A
Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, P.O. Box 210172, University of Cincinnati, Cincinnati, Ohio 45221, USA.
Borchers
Michael
M
Velayutham
Prakash
P
Wagner
Michael
M
Limbach
Patrick A
PA
D000171:Acrolein / Q000633:toxicity; D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry*; D019295:Computational Biology; D020869:Gene Expression Profiling; D008168:Lung / Q000187:drug effects; D051379:Mice; D040081:Protein Array Analysis / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011506:Proteins; D000171:Acrolein