Targeted proteomics using selected reaction monitoring (SRM)1
(also referred to as multiple reaction monitoring (MRM)) is a powerful technique that is widely used to quantify small molecules in complex matrices. More recently introduced in proteomics, it supports the identification and quantification of predetermined sets of peptides in complex samples, with a low limit of detection, wide dynamic range, high reproducibility and minimal redundancy (1
). For this technique, a specific mass spectrometric assay has to be developed once for each protein. Such assays are typically characterized by the identity of the analyte (i.e.
peptide amino acid sequence), the parent ion m
value, the approximate expected retention time of the targeted peptides, and the m
and relative signal intensity of product ions that are specifically associated with each precursor ion. These measures, if detected, uniquely identify the targeted peptide in a complex sample. The assays are generally optimized with respect to their fragmentation pattern with the background matrix of the sample origin (i.e.
plasma or cellular lysate). SRM assays can also be conducted using either native protein digests to detect targeted proteotypic peptides or can be incorporated in affinity capture routines such as N-glycocapture (3
) or immunoaffinity isolation (4
), to decrease complex digest solutions and increase both specificity and sensitivity to levels well within the pg/ml range (5
). Because these assays need to be generated only once per peptide and are increasingly publicly accessible in publications and databases, a generally accepted and transparent format for communicating SRM assays is a significant advance for this powerful targeted proteomics technology.
At present, a wide array of software tools are available to predict, select, validate and optimize transitions, such as TIQAM (6
), Skyline (7
), ATAQS (8
), as well as commercial offerings such as MRMPilot, Pinpoint, MassHunter, and VerifyE
, from AB SCIEX, Thermo Scientific, Agilent, and Waters, respectively. These tools use a variety of different, mostly tabular formats. Furthermore, emerging resources and tools for the generation and databasing of transitions such as PeptideAtlas (9
), SRMAtlas (11
), MRMaid (13
), MRMaid-DB (14
), GPMDB (15
), PASSEL (16
), and QuAD (http://proteome.moffitt.org/QUAD
) also support different formats.
The Human Proteome Organization (HUPO) Proteomics Standards Initiative (PSI; (17
)) has been instrumental in developing and supporting several standards for mass spectrometry data, including mzML (18
) for mass spectrometer output files and mzIdentML (20
) for the results of proteomics data processing. Each of the PSI formats is developed with similar concepts, such as controlled vocabularies and semantic validators. They follow a rigorous approval process that ensures that PSI formats are well tested and broadly applicable.
Toward unifying the fragmented state of SRM transition list formats, and facilitating communication between resources, tools, and instruments, the HUPO PSI Mass Spectrometry Standards Working Group has developed a new standardized format, TraML, that can be used to archive, share, and manage transition lists. In the following sections we describe the basic structure of the format, several use cases, and existing software implementations.