Solvent-dependent metabolite distribution, clustering, and protein extraction for serum profiling with mass spectrometry.
The aim of metabolite profiling is to monitor all metabolites within a biological sample for applications in basic biochemical research as well as pharmacokinetic studies and biomarker discovery. Here, novel data analysis software, XCMS, was used to monitor all metabolite features detected from an array of serum extraction methods, with application to metabolite profiling using electrospray liquid chromatography/mass spectrometry (ESI-LC/MS). The XCMS software enabled the comparison of methods with regard to reproducibility, the number and type of metabolite features detected, and the similarity of these features between different extraction methods. Extraction efficiency with regard to metabolite feature hydrophobicity was examined through the generation of unique feature density distribution plots, displaying feature distribution along chromatographic time. Hierarchical clustering was performed to highlight similarities in the metabolite features observed between the extraction methods. Protein extraction efficiency was determined using the Bradford assay, and the residual proteins were identified using nano-LC/MS/MS. Additionally, the identification of four of the most intensely ionized serum metabolites using FTMS and tandem mass spectrometry was reported. The extraction methods, ranging from organic solvents and acids to heat denaturation, varied widely in both protein removal efficiency and the number of mass spectral features detected. Methanol protein precipitation followed by centrifugation was found to be the most effective, straightforward, and reproducible approach, resulting in serum extracts containing over 2000 detected metabolite features and less than 2% residual protein. Interestingly, the combination of all approaches produced over 10,000 unique metabolite features, a number that is indicative of the complexity of the human metabolome and the potential of metabolomics in biomarker discovery.
Want
Elizabeth J
EJ
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
O'Maille
Grace
G
Smith
Colin A
CA
Brandon
Theodore R
TR
Uritboonthai
Wilasinee
W
Qin
Chuan
C
Trauger
Sunia A
SA
Siuzdak
Gary
G
D001798:Blood Proteins / Q000032:analysis* / Q000302:isolation & purification; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D008297:Male; D012680:Sensitivity and Specificity; D044967:Serum / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D001798:Blood Proteins; D012997:Solvents