Between‐gel reproducibility of the human cerebrospinal fluid proteome

Abstract

This manuscript describes the between-gel reproducibility of the two-dimensional gel electrophoresis analysis of the human lumbar cerebrospinal fluid (CSF) proteome. This reproducibility study is a necessary component for our long-term research program that uses comparative proteomics to analyze lumbar CSF samples in a study of human idiopathic low back pain. A Protein-Plus Dodeca Cell electrophoresis apparatus and PDQuest software were used to measure the level of between-gel reproducibility of the CSF proteome. One pooled CSF sample was used to evaluate the level of within-sample, between-gel reproducibility, and a set of seven different CSF samples (CSF-1 to 7) was used to test the level of within-group and between-group variability. Differentially expressed proteins (six CSF samples versus the designated control, CSF-3) were characterized with mass spectrometry. The number of spots found in the pooled CSF sample was 490 ± 30 (n = 10 gels); the percentage of protein spots found in those 10 gels was 92 ± 6%, with a coefficient of variation of 6%; and a positive coefficient of correlation (r = 0.82) was found. In order to test the proof-of-principle, that set of seven CSF samples served as a test of our ability to perform reproducibility comparative proteomics, and to detect differentially expressed proteins within that set of test samples. One sample (CSF-3) served as the control for the other six to locate the differentially expressed proteins. A comparison of fifteen differentially expressed proteins found in that set of test CSF samples correlated with pathology. Matrix-assisted laser desorption/ionization-time-of-flight and electrospray ionization quadrupole ion trap mass spectrometry were used to characterize thirteen of those fifteen differentially expressed proteins. These results (reproducibility, protein characterization, set of test samples, and proof-of-principle) suggest that the analysis of human CSF two-dimensional gels can achieve a high level of within-sample and between-sample reproducibility, and that PDQuest software can measure the relative protein abundance in the human CSF proteome.