Shotgun proteomics is named for its similarity to shotgun sequencing, a method used in genomic sequencing from 1995-2005, which has since been replaced by next-generation technologies.1 Given the rapid advancement in technology for molecular biology and that shotgun proteomics has been around for more than 10 years, one might ask if this technology is now becoming outdated.
Shotgun proteomics has previously been described as “…the most powerful and comprehensive technique for mapping out the proteome”.2 First, the proteins are extracted from a biological sample and then digested. From there, reverse phase liquid chromatography or electrophoresis can be used to fractionate the resulting peptides. Next, tandem mass spectrometry (MS) is performed on the sample and the results are matched to previously known peptides.3
While this high-throughput approach enables researchers to analyze multiple proteins that may be present in a sample, confusion can arise if the resulting peptides end up matching ambiguously to multiple peptides in the database. This can happen in especially complex samples, such as infectious disease samples that can contain proteins from multiple organisms. To get around this issue, a number of analysis methods and statistical algorithms have been used to confidently validate and reconstruct proteins.4 Researchers are also looking at ways to enhance the identification of proteins during MS. It is estimated that the majority of proteins detected are currently inaccessible due to limitations with traditional mass spectrometry, and strategies are being developed to more fully understand how to maximize MS coverage.5
Shotgun proteomics methods are being paired with other techniques to gain a more complete picture of environmental changes in phosphate deficiency (Pi). Using shotgun proteomics, as well as whole-genome RNA sequencing, the Lan group investigated correlations between Pi deficiency-induced changes in transcriptome and proteome profiles in Arabidopsis roots and was able to reliably identify 13,298 proteins and 24,591 transcripts, subsets of 356 proteins and 3,106 mRNAs differentially expressed during Pi deficiency. They also noted that in several cases, upregulation of gene activity was observed solely at the protein level, and the information added novel aspects to key processes in the adaptation to Pi deficiency.6
Investigations using shotgun proteomics are now being adapted to areas of science that were previously dominated by DNA assays. In an article published in July 2012, Corthals et al. report on their new use of shotgun proteomics to detect disease in a 500 year-old Incan mummy. This type of work had never been done before and offers some distinct advantages over traditional methods. In the past, DNA assays could only detect the presence of pathogens and not infer disease since pathogens can be present without causing disease. Proteomics offers the ability to characterize multiple proteins simultaneously with less susceptibility to contamination than with PCR amplification.7
According to the PubMed database, there were 735 articles published from Jan 1, 2011 to Dec 31, 2012 using shotgun proteomics, with 212 of those articles published in 2011.8 As research techniques are further enhanced, new applications for shotgun proteomics will likely be found.
3. Marcotte, E.M. (2007) ‘How do shotgun proteomics algorithms identify proteins?‘, Nature Biotechnology, 25 (7), (pp. 755-757)
4. Shen, C., et al. (2008) ‘A hierarchical statistical model to assess the confidence of peptides and proteins inferred from tandem mass spectrometry‘, Bioinformatics, 24 (2), (pp 202-208)
5. Michalski, A., Cox J., and Mann M. (2011) ‘More than 100,000 detectable peptide species elute in single shotgun proteomics runs but the majority is inaccessible to data-dependent LC-MS/MS‘, Journal of Proteome Research, 10 (4), (pp. 1785-1793)
6. Lan, P., Li, W., and Schmidt, W. (2012) ‘Complementary Proteome and Transcriptome Profiling in Phosphate-Deficient Arabidopsis Roots Reveals Multiple Levels of Gene Regulation.’ Molecular and Cellular Proteomics, published July 25, 2012. doi: 10.1074/mcp.M112.020461
7. Corthals, A., et al. (2012) ‘Detecting the immune system response of a 500-year-old Incan mummy‘, published online July 25, 2012. doi: 10.1371/journal.pone.0041244
8. National Center for Biotechnology Information, PubMed database, http://www.ncbi.nlm.nih.gov/pubmed