Reproducibility of retention time using a splitless nanoLC coupled to an ESI-FTICR mass spectrometer.
Replicate injections of a myoglobin tryptic digest, ultrafiltrates of human serum, and ultrafiltrates of human plasma made on a splitless nanoscale liquid chromatography system coupled to a Fourier-transform ion cyclotron resonance mass spectrometer were utilized to assess analytical reproducibility. The mean (across 19 tryptic fragments detected in at least 3 of 24 replicate injections) of the 95% CIM of retention time is +/-6.3 sec and the maximum is +/-11.6 sec; when only those tryptic fragments that were found in 24 of 24 replicates are considered, the maximum 95% CIM of retention time drops to +/-6.7 sec. This represents a deviation of at most seven spectra. Similarly, in the serum (and plasma) filtrates, 95% of the 393 (312) species observed in 3 replicate injections had a 95% CIM of retention time of +/-22.0 (+/-18.5) sec or less. Ion abundance was similarly reproducible, with an average across those tryptic fragments observed in all 24 replicates of the coefficient of variation of ion abundance equal to 37.0%. This reproducibility represents a significant improvement over prior work, which required flow splitting in order to achieve nanoliter-per-minute flow rates. These improvements in retention time reproducibility will also be observed with mass spectrometers employing mass analyzers other than FT-ICR.
Mason
Christopher J
CJ
WM Keck FT-ICR Mass Spectrometry Laboratory, Mayo Proteomics Research Centers, Rochester, Minnesota, USA.
Johnson
Kenneth L
KL
Muddiman
David C
DC
D016022:Case-Control Studies; D002853:Chromatography, Liquid; D005260:Female; D005583:Fourier Analysis; D006801:Humans; D013058:Mass Spectrometry; D008875:Middle Aged; D036103:Nanotechnology / Q000379:methods*; D009367:Neoplasm Staging; D010051:Ovarian Neoplasms / Q000097:blood / Q000473:pathology; D017698:Postmenopause; D015203:Reproducibility of Results; D012984:Software; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors