Targeted peptide measurements in biology and medicine: best practices for mass spectrometry-based assay development using a fit-for-purpose approach.
Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Publications describing targeted MS assays for peptides frequently do not contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs. Guidance must be developed so that targeted MS assays with established performance can be made widely distributed and applied by many labs worldwide. To begin to address the problems and their solutions, a workshop was held at the National Institutes of Health with representatives from the multiple communities developing and employing targeted MS assays. Participants discussed the analytical goals of their experiments and the experimental evidence needed to establish that the assays they develop work as intended and are achieving the required levels of performance. Using this "fit-for-purpose" approach, the group defined three tiers of assays distinguished by their performance and extent of analytical characterization. Computational and statistical tools useful for the analysis of targeted MS results were described. Participants also detailed the information that authors need to provide in their manuscripts to enable reviewers and readers to clearly understand what procedures were performed and to evaluate the reliability of the peptide or protein quantification measurements reported. This paper presents a summary of the meeting and recommendations.
Broad Institute of MIT and Harvard, Cambridge, Massachusetts;
Steven A
SA
Carr
Susan E
SE
Abbatiello
Bradley L
BL
Ackermann
Christoph
C
Borchers
Bruno
B
Domon
Eric W
EW
Deutsch
Russell P
RP
Grant
Andrew N
AN
Hoofnagle
Ruth
R
Hüttenhain
John M
JM
Koomen
Daniel C
DC
Liebler
Tao
T
Liu
Brendan
B
MacLean
D R
DR
Mani
Elizabeth
E
Mansfield
Hendrik
H
Neubert
Amanda G
AG
Paulovich
Lukas
L
Reiter
Olga
O
Vitek
Ruedi
R
Aebersold
Leigh
L
Anderson
Robert
R
Bethem
Josip
J
Blonder
Emily
E
Boja
Julianne
J
Botelho
Michael
M
Boyne
Ralph A
RA
Bradshaw
Alma L
AL
Burlingame
Daniel
D
Chan
Hasmik
H
Keshishian
Eric
E
Kuhn
Christopher
C
Kinsinger
Jerry S H
JS
Lee
Sang-Won
SW
Lee
Robert
R
Moritz
Juan
J
Oses-Prieto
Nader
N
Rifai
James
J
Ritchie
Henry
H
Rodriguez
Pothur R
PR
Srinivas
R Reid
RR
Townsend
Jennifer
J
Van Eyk
Gordon
G
Whiteley
Arun
A
Wiita
Susan
S
Weintraub
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D001695:Biology; D017408:Guidelines as Topic; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D008511:Medicine; D010455:Peptides / Q000378:metabolism*; D040901:Proteomics / Q000592:standards; D012015:Reference Standards; D012984:Software
D010455:Peptides