Reproducibility of tryptic digestion investigated by quantitative fourier transform ion cyclotron resonance mass spectrometry.
In this study, the reproducibility of tryptic digestion of complex solutions was investigated using liquid chromatography Fourier transform ion cyclotron resonance (LC FT-ICR) mass spectrometry. Tryptic peptides, from human cerebrospinal fluid, (CSF) were labeled with Quantification-Using-Enhanced-Signal-Tags (QUEST)-markers, or 1-([H4]nicotinoyloxy)- and 1-([D4]nicotinoyloxy)-succinimide ester markers. The analysis was performed on abundant proteins with respect-to-intensity ratios and sequence coverage and obtained by comparing differently labeled components from one or different pools. To interpret the dynamics in the proteome, one must be able to estimate the error introduced in each experimental steps. The intra sample variation due to derivatization was approximately 10%. The inter sample variation depending on derivatization and tryptic digestion was not more than approximately 30%. These experimental observations provide a range for the up- and down-regulations that are possible to study with electrospray ionization LC FT-ICR mass spectrometry.
Hagman
Charlotte
C
Division of Ion Physics, The Angström Laboratory, Uppsala University, Box 534, SE-75121 Uppsala, Sweden.
Ramström
Margareta
M
Jansson
Maria
M
James
Peter
P
Håkansson
Per
P
Bergquist
Jonas
J
D000293:Adolescent; D000328:Adult; D000368:Aged; D002851:Chromatography, High Pressure Liquid; D017357:Cyclotrons; D005583:Fourier Analysis; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D010455:Peptides / Q000134:cerebrospinal fluid*; D020543:Proteome; D015203:Reproducibility of Results; D014357:Trypsin / Q000737:chemistry*
D010455:Peptides; D020543:Proteome; D014357:Trypsin