One of the critical decisions one has to make during SRM method development is to select transitions for monitoring peptides. In practice, the choice of transitions is often based on the observed signal intensity and chromatographic performance. We recommend, however, that at the beginning of method development one do a screening SRM in the matrix of interest (e.g. serum, plasma) involving the theoretical transitions listed in the deliverable .csv regardless of ion redundancy hit scores. At the end of the screening experiment, one can narrow down the transitions by eliminating those that did not produce quantifiable signals. Ideally, among the remaining transitions that produced quantifiable signals there exist unique transitions carrying redundancy hit score of 1. These unique transitions should be advanced to the next stage of method development. In cases where unique transitions are not identified (not listed in the deliverable .csv or eliminated during screening experiment), one should prioritize transitions in favor of those with lower redundancy hit scores. If needed, one can use synthetic peptides under exploratory chromatographic conditions to confirm whether the reported redundant ions actually co-elute with the target ions as the calculated retention time may not be accurate in our redundancy evaluation. Furthermore, using synthetic or proteolytic peptides, one may be able to experiment and identify a unique LC condition (not necessarily reversed-phase LC) to separate the redundant ions listed in our report, which is not possible without knowing what the redundant ions are in the first place.

We implemented published methods (Tripet, B., et al. J Chromatogr A 2007 1141(2): 212-225; Petritis, K., et al. Anal Chem 2006 78(14): 5026-5039) to calculate peptide retention time. Peptides are considered co-eluting if the calculated retention times fall within +/- 12 seconds. This criteria has been optimized to support typically short runs of production SRM assays (e.g. the vast majority of validated SRM methods are under 15 minutes in support of clinical studies).

The criteria for defining indistinguishable ions is +/- 0.5 amu, which is the typical mass spectral resolution on today’s triple quadrupole mass spectrometers. The same criteria is applied to both precursor and product ions.

In theory, there exist other types of sequence ions. In practice, however, the singly charged y and b ions tend to dominate the product ion landscape. Because of this fragmentation behavior, we focus on the singly charged y and b ions for redundancy evaluation.

• peptide length: 5-20 amino acids
• enzymatic digestion: fully digested
• peptide modification: no modification (e.g. phosphorylation) considered
• peptide precursor charge: doubly charged (tryptic)
• product ion charge: singly charged y and b only
• co-eluting: +/- 12 seconds in calculated retention time
• indistinguishable ion: +/- 0.5 amu (both precursor and product ions)

Yes, we support other species in addition to clinical and preclinical model organisms (e.g. yeast, fruit fly, C. elegans, Zebrafish, frog, Arabidopsis, etc.). In other words, we can support any species that is represented in the UniProtKB database. Please let us know if the species of your interest is not listed in the “Place An Order” section on our website.

Yes, we can support other enzymes in addition to Trypsin for protein digestion (e.g. Chymotrypsin or Lys-C). We focused on Trypsin because Trypsin is the most commonly used enzyme in bottom-up quantitative proteomics. Please let us know if the amino acid sequence of your target protein requires proteolysis by Chymotrypsin or Lys-C.

If your target protein is not in the UniProtKB database (e.g. a recombinant protein), you will need to provide the amino acid sequence for us to perform the ion redundancy evaluation.

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