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ISI Journal Citation Reports © Ranking: 2012: 14/75 (BIOCHEMICAL RESEARCH METHODS); 78/290 (Biochemistry & Molecular Biology)
Online ISSN: 1615-9861
Associated Title(s): PROTEOMICS - Clinical Applications
Comments to the Viewpoint article
How specific is my SRM?: The issue of precursor and product ion redundancy
Jamie Sherman, Matthew J. McKay, Keith Ashman, Mark P. Molloy
Published Online: Feb 27 2009
|Comment from:||Rastislav Sramek|
|Short affiliation:||Institute for Theoretical Computer Science, ETH Zurich|
|e-mail:||email@example.com Date: 4 October 2010|
Having stumbled upon this paper only now, I'd like to point out related work we have done earlier, that might possibly of some interest: http://dx.doi.org/10.1007/978-3-642-02882-3_29
|Comment from:||Dr. Metodi V. Metodiev|
|Short affiliation:||Proteomics Unit, University of Essex, UK|
|e-mail:||firstname.lastname@example.org Date: 20 May 2009|
The two viewpoint papers on peptide SRM by Sherman et al. and Duncan et al. are timely and rise very important questions. Simply put, the gravest problems seem to come from the fact that peptide/protein quantitation by SRM relies on the conventional form of the technique that has been established and used for small organic molecules, not proteolytic peptides. With proteome samples the so called analytical space is much greater. Hence there is significant probability that a SRM transition used to measure a peptide with a given sequence would be generated by a peptide with a different sequence. This is clearly illustrated by the occurrence of multiple peaks in the SRM traces when analysing peptides. The argument that retention time would provide additional dimension, thus increasing specificity, is a bit shaky too as peptides with the same amino acid composition but different sequence (scrambled peptides that might still give rise to the same SRM transitions) would be expected to have very similar retention times. It is therefore quite serious analytical problem that may need a radical approach to be solved. In my lab we are trying to overcome this problem by introducing MS3 SRM. We believe that peptides require this extra step for specificity. We use ion traps to do this, which will certainly prompt hard-core triple quad folks to say that this cannot be quantitative enough. However, with the fast scanning ion trap instruments available today, such a statement is not necessarily true. What is more serious as a limitation of this MS3 SRM is the inevitable loss of signal intensity. This would obviously translate into loss of sensitivity, but maybe it is a price we should pay if we want ultimate specificity of our peptide quantitation. There might be an additional advantage to use an extra step in SRM. Since MS3 SRM is much more specific (only single peaks are seen in the SRM trace) there is no need to use labelled internal standards and the requirement for very high reproducibility of the retention times is not essential. An additional rationale to use MS3 is the complicated case of peptide phosphorylation. In fact we never attempt MS2 SRM on peptides phosphorylated on serine or threonine. The yield of useful fragments is very low due to the neutral loss phenomenon. This is now a well established fact and data-dependent MS3 is routinely used to map phosphorylation sites in an automatic way.
|Comment from:||Dr. Jie Li|
|Short affiliation:||Institute of Genetics and Developmental Biology, Beijing, China|
|e-mail:||email@example.com Date: 12 May 2009|
Each protein quantification method has its inherent pros and cons. With the widening application of LC-MS/MS in peptide quantification, the pros and cons of the method need to be known and considered when analyzing the results. The articles by Sherman et al. and Duncan et al. describe the various aspects of the SRM.
|Comment from:||Richard Kay|
|Short affiliation:||Quotient Bioresearch Ltd, Cambridgeshire, UK|
|e-mail:||firstname.lastname@example.org Date: 08 May 2009|
I work in an analytical chemistry laboratory where we develop and validate LC-MS/MS and SRM based methods for peptide and small molecule analytes to GLP standards. I found the articles by Sherman and Duncan to be extremely interesting and posed some interesting problems that researchers looking to validate SRM based protein quantitative methods need to address. I believe these questions can be partly answered by adopting selected procedures from existing bioanalytical method validation processes such as those outlined by Viswanathan et al. (Pharmaceutical Research Vol. 24, No. 10, October 2007). Furthermore, validation of an LC-MS/MS and SRM based method for a tryptic peptide surrogate should also include evidence that the peptide is derived from the intact protein in question (selectivity to intact protein).
|Comment from:||Dr. Rachel Ogorzalek Loo|
|Short affiliation:||Laboratory for Proteomics and Genomics, LA University of California, USA|
|e-mail:||email@example.com Date: 07 May 2009|
The excellent articles by Sherman et al. and Duncan et al. raise important points. Additionally, sequence variants present in the population must be considered, and for an individual whether they are homozygous or heterozygous for a particular peptide. A look at Swiss Prot's human albumin entry provides a daunting view of the variants presently characterized. As more and more data is obtained on human haplotypes (variants present in >1% of the population), their impact will have to be considered.
|Comment from:||Professor Stephen Barnes|
|Short affiliation:||Targeted Metabolomics and Proteomics Laboratory, University of Alabama at Birmingham, Birmingham, AL, USA|
|e-mail:||firstname.lastname@example.org Date: 06 May 2009|
MRM is of big interest - we've been using this approach for small molecules and now peptides for several years. However, like everything else in analytical biochemistry, it's not a cure all, but as long as you're aware of its limitations, it shouldn't be a problem. The key is to use an instrument like a Sciex 5500 Qtrap, where because of its sensitivity and speed, you can get a meaningful and confirmatory MS/MS spectra during the capture of specific parent ion/daughter ion transitions.
|Comment from:||Dr. Yu-Jen Wu and Chih-Ming Lu|
|Short affiliation:||Meiho Institute of Technology, Pingtung, Taiwan|
|e-mail:||email@example.com Date: 06 May 2009|
|Comment:||We mainly agree with the viewpoint presented by Sherman et al. Quantification of a target peptide from a complex sample only using SRM technique is not sufficient. Redundancy on both precursor and product ion may lead to an ambiguous quantification or even a false result. My knowledge about SRM is that usually a target standard (usually a synthetic peptide) is required to fix the retention time of liquid chromatography, to provide the fragmentation details, and to generate a calibration curve. The redundancy in SRM experiments could be narrowed down to a minimum as the possibility to encounter a redundant precursor with several identical product ions (instead of one single product ion) with the same retention time is very low. Nevertheless, we still can not rule out the possible redundancy. Therefore, to obtain a convincing data for a proteome research, we may need at least two independent analytical methods to validate with each other. Alternative methods such as stable isotope labeling or western blotting experiment may be required to confirm the result obtained by SRM.|
|Comment from:||Dr. Richard Unwin|
|Short affiliation:||Stem Cell & Leukaemia Proteomics Laboratory, University of Manchester, UK|
|e-mail:||firstname.lastname@example.org Date: 05 May 2009|
|Comment:||The articles by Sherman et al. and Duncan et al. raise valid points about the use of SRM for protein quantification, and both provide warnings of what might go wrong with this kind of analysis.|
Sherman et al. are correct in their statement that a peptide SRM is, in fact, defined by three factors, namely peptide m/z, fragment m/z and retention time. Performing SRM-triggered MS/MS of all target peptides to ensure 'specificity' is a prerequisite, as is sufficiently high quality sample preparation and pre-fractionation/chromatography to minin=mise potential problems.
However, many of these issues are solved by careful selection of peptides. Database searching for tryptic peptides of similar mass and determination of their fragment ion masses should enable optimum design of SRM pairs, when coupled with the above.
Duncan et al. do, however, raise a more pressing point regarding peptide post translational modifications, especially as a given protein may exist in many forms, each with different combinations of PTM. Realistically, there is not much that the experimentalist can do about this, other than be aware of known PTMs on their target of interest and try to avoid them. Avoiding all peptides which contain one of M, C, S, T or Y (even before you begin to consider deamidation occurring during sample prep, glycosylation etc) is impractical. Also the number of peptides required to accurately assess the quantity of a protein is important. Good practice dictates that using several peptides from a protein is the best way forward (although to play devils advocate, is using one peptide any worse than the current gold standard, namely using an antibody which recognises a single epitope?). However, the use of several peptides does allow either an 'average' to be determined, or outliers (which may occur as a result of PTM) to be identified.
In summary these articles provide a good assessment of the factors which require consideration while designing good SRM-based experiments, and the results of these considerations should be made available as supplementary data when such an analysis is published.
|Comment from:||Professor Ian Humphery-Smith|
|Short affiliation:||Newcastle-upon-Tyne, UK|
|e-mail:||email@example.com Date: 04 May 2009|
|Comment:||I read both articles and thought the forum of opinions by experts on evolving / emerging techniques was EXCELLENT innovation. It would be nice to see more of same vein....|
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