Nanoflow LC-MS for High-Performance Chemical Isotope Labeling Quantitative Metabolomics.
Nanoflow liquid chromatography mass spectrometry (nLC-MS) is frequently used in the proteomics field to analyze a small amount of protein and peptide samples. However, this technique is currently not widespread in the metabolomics field. We report a detailed investigation on the development of an nLC-MS system equipped with a trap column for high-performance chemical isotope labeling (CIL) metabolomic profiling with deep coverage and high sensitivity. Experimental conditions were optimized for profiling the amine/phenol submetabolome with (13)C-/(12)C-dansylation labeling. Comparison of analytical results from nLC-MS and microbore LC-MS (mLC-MS) was made in the analysis of metabolite standards and labeled human urine and sweat samples. It is shown that, with a 5-μL loop injection, 7 labeled amino acid standards could be detected with S/N ranging from 7 to 150 by nLC-MS with an injection of 5 nM solution containing a total of 25 fmol labeled analyte. For urine metabolome profiling where the sample amount was not limited, nLC-MS detected 13% more metabolites than mLC-MS under optimal conditions (i.e., 4524 ± 37 peak pairs from 26 nmol injection in triplicate vs 4019 ± 40 peak pairs from 52 nmol injection). This gain was attributed to the increased dynamic range of peak detection in nLC-MS. In the analysis of human sweat where the sample amount could be limited, nLC-MS offered the advantage of providing much higher coverage than mLC-MS. Injecting 5 nmol of dansylated sweat, 3908 ± 62 peak pairs or metabolites were detected by nLC-MS, while only 1064 ± 6 peak pairs were detected by mLC-MS. Because dansyl labeled metabolites can be captured on a reversed phase (RP) trap column for large volume injection and are well separated by RPLC, the CIL platform can be readily implemented in existing nLC-MS instruments such as those widely used in shotgun proteomics.
Department of Chemistry, University of Alberta , Edmonton, Alberta T6G, Canada.
Zhendong
Z
Li
Department of Chemistry, University of Alberta , Edmonton, Alberta T6G, Canada.
Jaspaul
J
Tatlay
Department of Chemistry, University of Alberta , Edmonton, Alberta T6G, Canada.
Liang
L
Li
D000588:Amines / Q000032:analysis* / Q000378:metabolism; D002247:Carbon Isotopes; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation*; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000295:instrumentation*; D055432:Metabolomics / Q000379:methods*; D036103:Nanotechnology / Q000295:instrumentation*; D010636:Phenols / Q000032:analysis* / Q000378:metabolism
D000588:Amines; D002247:Carbon Isotopes; D010636:Phenols