Repeatability and reproducibility in proteomic identifications by liquid chromatography-tandem mass spectrometry.
The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35-60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies.
Tabb
David L
DL
Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
Vega-Montoto
Lorenzo
L
Rudnick
Paul A
PA
Variyath
Asokan Mulayath
AM
Ham
Amy-Joan L
AJ
Bunk
David M
DM
Kilpatrick
Lisa E
LE
Billheimer
Dean D
DD
Blackman
Ronald K
RK
Cardasis
Helene L
HL
Carr
Steven A
SA
Clauser
Karl R
KR
Jaffe
Jacob D
JD
Kowalski
Kevin A
KA
Neubert
Thomas A
TA
Regnier
Fred E
FE
Schilling
Birgit
B
Tegeler
Tony J
TJ
Wang
Mu
M
Wang
Pei
P
Whiteaker
Jeffrey R
JR
Zimmerman
Lisa J
LJ
Fisher
Susan J
SJ
Gibson
Bradford W
BW
Kinsinger
Christopher R
CR
Mesri
Mehdi
M
Rodriguez
Henry
H
Stein
Stephen E
SE
Tempst
Paul
P
Paulovich
Amanda G
AG
Liebler
Daniel C
DC
Spiegelman
Cliff
C
D002853:Chromatography, Liquid / Q000379:methods*; D020543:Proteome; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
D020543:Proteome