alexa MRM Proteomics | Quantification | Sensitivity
ISSN: 2155-9872
Journal of Analytical & Bioanalytical Techniques
Like us on:
Make the best use of Scientific Research and information from our 700+ peer reviewed, Open Access Journals that operates with the help of 50,000+ Editorial Board Members and esteemed reviewers and 1000+ Scientific associations in Medical, Clinical, Pharmaceutical, Engineering, Technology and Management Fields.
Meet Inspiring Speakers and Experts at our 3000+ Global Conferenceseries Events with over 600+ Conferences, 1200+ Symposiums and 1200+ Workshops on
Medical, Pharma, Engineering, Science, Technology and Business

Targeted MRM Proteomics is a Better Protein Quantification Method Over Western-Blotting

Bhagwat Prasad*

Department of Pharmaceutics, University of Washington, USA

*Corresponding Author:
Bhagwat Prasad
Department of Pharmaceutics, University of Washington
Seattle, P.O. Box 357610, WA 98195, USA
Tel: +1-206-221-3035
Fax: +1-206-543-3204
E-mail: [email protected]

Received date: October 24, 2013; Accepted date: October 25, 2013; Published date: October 28, 2013

Citation: Prasad B (2013) Targeted MRM Proteomics is a Better Protein Quantification Method Over Western-Blotting. J Anal Bioanal Tech 4:e117. doi: 10.4172/2155-9872.1000e117

Copyright: © 2013 Prasad B. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Visit for more related articles at Journal of Analytical & Bioanalytical Techniques

Keywords

Sensitivity and selectivity ofliquid chromatographycoupled with tandem mass spectrometry (LC-MS/MS) for quantification of small molecules encouraged biologists to utilize this platform for protein quantification using peptide as a surrogate. MS based protein quantification, commonly referred as multiple or selective reaction monitoring (MRM or SRM) proteomics, relies on selective quantification of surrogate peptide(s) in a digested protein sample. The selectivity is achieved by the MRM characteristic of triple quandrupole MS instrument in which unique daughter ion generated from fragmentation of a parent ion (i.e, ionized analyte peptide) is monitored. This application of MS based targeted proteomics become wider with the availability of protein databases and in silico methods for surrogate peptide selection in the last five years. For instance, successful completion of human genome project and the ongoing human proteome project enabled us to select surrogate peptides for MRM quantification of nearly every human protein [1,2]. Because of its increasing popularity, Nature Methods declared targeted proteomics by mass spectrometry (MS) as Method of the Year for 2012 [3].

On the other hand, western-blotting (or protein immunoblotting) is a conventional protein quantification method which is a standard technique in almost every biology laboratory. Therefore, it is a challenge for biologists to see beyond western-blotting, and accept and adopt MRM proteomics in routine protein quantification work. Considering the latter, it is important to compare the advantages and disadvantages of both these methods. Aebersold et al. [4] recently recently touched this topic in their editorial in Molecular &Cellular Proteomics and advocated the use of MRM proteomics. As a user, I present here my perspective on this topic focusing on the differences between the two methods on various aspects, i.e., application and speed, ease of use, quality of data and ability to confirm results. i) MRM proteomics can be used to quantify multiple proteins in a shorter time (approximately 10 proteins in 20 min) [5]. As the latter does not rely on antibodies, it can quantify every protein for which the sequence is known. On the other hand, western-blotting is mostly applicable to one protein at a time and requires antibodies. We even use MRM to quantify complex membrane bound proteins for which developing good antibodies is a huge challenge [5-9]. ii) Western-blotting is recognized as a routine method and can be applied to any laboratory; however, MRM needs sophisticated MS instrument with expertise to run the same. Despite that, any laboratory that is using MRM for quantification of small molecules, can adopt this technology in protein quantification. Further, with the availability of in silico tools, it takes approximately 1 hour to select a surrogate peptide and confirm the signal in a digested biological sample using MRM method. iii) As synthetic peptide is used as calibrator, MRM proteomics can deliver absolute protein levels when the protein digestion efficiency is established. Sensitivity of MRM method depends on peptide characteristics and may vary widely. In general, if the best peptide is used for MRM quantification, it is comparable or better in sensitivity than western-blotting. This is because signal to noise ratio is significantly high and a minimal sample volume is required in MRM. The MRM sensitivity can be further increased substantially by using stable isotope standard capture with anti-peptide antibodies (SISCAPA) [10,11]. As protein digestion is reproducible and many sample preparation steps can be automated, MRM offers better precision. iv) The quality of data is also better in the latter because it rely on multiple signals, i.e., multiple peptides and multiple MRM transitions. The results can be easily confirmed by using another independent surrogate peptide.

So, LC-MRM outperforms western-blotting in many aspects except that a dedicated facility with skilled personnel are required to run these sophisticated instruments and therefore the initial costs are higher. However, with the pace of technology, it is not difficult to imagine that the small, economical and user-friendly bench-top LCMRM instruments will be available soon. The main advantage of LCMRM is its ability to quantify various proteins in multiple samples with great high-throughput. This is particularly applicable in protein quantification in biofluids, tissues, cells and cell lines for applications like, biomarker discovery and validation, patient stratification, clinical trial monitoring, development of companion diagnostics, drug toxicity assessments in serum or urine samples, genomics-proteomics correlations, systems biology/pharmacology (network analysis), systems based pharmacokinetic (SBPK) modeling, pharmacokineticspharmacodynamic (PKPD) modeling, pharmacokinetic (PK) studies of bio-therapeutics, population variability in protein expression, quality control studies of biologics.

References

Select your language of interest to view the total content in your interested language
Post your comment

Share This Article

Relevant Topics

Recommended Conferences

Article Usage

  • Total views: 11973
  • [From(publication date):
    February-2014 - Aug 17, 2017]
  • Breakdown by view type
  • HTML page views : 8168
  • PDF downloads :3805
 

Post your comment

captcha   Reload  Can't read the image? click here to refresh

Peer Reviewed Journals
 
Make the best use of Scientific Research and information from our 700 + peer reviewed, Open Access Journals
International Conferences 2017-18
 
Meet Inspiring Speakers and Experts at our 3000+ Global Annual Meetings

Contact Us

 
© 2008-2017 OMICS International - Open Access Publisher. Best viewed in Mozilla Firefox | Google Chrome | Above IE 7.0 version
adwords