Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry. II. Limitations of complex mixture analyses.
With an emphasis on obtaining a multitude of high quality tandem mass spectrometry spectra for protein identification, instrumental parameters are described for the liquid chromatography-tandem mass spectrometry analysis of trypsin digested unfractionated urine using a hybrid quadrupole-time-of-flight (Q-TOF) mass spectrometer. Precursor acquisition rates of up to 20 distinct precursors/minute in a single analysis were obtained through the use of parallel precursor selection (four precursors/survey period) and variable collision induced dissociation integration time (1 to 6 periods summed). Maximal exploitation of the gas phase fractionated ions was obtained through the use of narrow survey scans and iterative data-dependent analyses incorporating dynamic exclusion. The impact on data fidelity as a product of data-dependent selection of precursor ions from a dynamically excluded field is discussed with regards to sample complexity, precursor selection rates, survey scan range and facile chemical modifications. Operational and post-analysis strategies are presented to restore data confidence and reconcile the greatest number of matched spectra.
Davis
M T
MT
Department of Biochemistry and Genetics, Amgen, Thousand Oaks, CA, USA.
Spahr
C S
CS
McGinley
M D
MD
Robinson
J H
JH
Bures
E J
EJ
Beierle
J
J
Mort
J
J
Yu
W
W
Luethy
R
R
Patterson
S D
SD
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D020543:Proteome / Q000235:genetics / Q000302:isolation & purification*; D014556:Urine / Q000737:chemistry
D010446:Peptide Fragments; D020543:Proteome