Abstract
A crucial part of optimization of metabolically engineered organisms is producing balanced levels of pathway proteins. Typically, protein levels are monitored by Western blot analysis; however, application to multiple enzyme pathways can be difficult without unique antibodies for each enzyme in the pathway. Furthermore, it can be time consuming, and cost prohibitive during exploratory stages of pathway design when many different proteins must be monitored simultaneously. We present here a targeted proteomics approach that uses selected-reaction monitoring (SRM) mass spectrometry to quantify multiple proteins in a sample. SRM methods provide high selectivity and high sensitivity to enable rapid quantification of multiple proteins in an engineered pathway regardless of sequence or organism of origin.
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References
Lange V, Picotti P, Domon B, Aebersold R (2008) Selected reaction monitoring for quantitative proteomics: a tutorial. Mol Syst Biol 4:222
Baillie TA (2008) Metabolism and toxicity of drugs. Two decades of progress in industrial drug metabolism. Chem Res Toxicol 21(1):129–137
Picotti P, Rinner O, Stallmach R et al (2010) High-throughput generation of selected reaction-monitoring assays for proteins and proteomes. Nat Methods 7(1):43–46
Deutsch EW, Lam H, Aebersold R (2008) PeptideAtlas: a resource for target selection for emerging targeted proteomics workflows. EMBO Rep 9(5):429–434
Wessel D, Flügge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138:141–143
Sun W, Gao S, Wang L et al (2006) Microwave-assisted Protein Preparation and Enzymatic Digestion in Proteomics. Mol Cell Proteomics 5(4):769–776
Yeung Y-G, Nieves E, Angeletti RH, Stanley ER (2008) Removal of detergents from protein digests for mass spectrometry analysis. Anal Biochem 382(2):135–137
Bluemlein K, Ralser M (2011) Monitoring protein expression in whole-cell extracts by targeted label- and standard-free LC-MS/MS. Nat Protoc 6(6):859–869
Hunt DF, Yates DR, Shabanowitz J et al (1986) Protein sequencing by tandem mass spectrometry. Proc Natl Acad Sci USA 83:6233–6237
Prakash A, Tomazela DM, Frewen B et al (2009) Expediting the development of targeted SRM assays: using data from shotgun proteomics to automate method development. J Proteome Res 8(6):2733–2739
Papayannopoulos IA (1995) The interpretation of collision-induced dissociation tandem mass spectra of peptides. Mass Spectrom Rev 14:49–73
Picotti P, Lam H, Campbell D et al (2008) A database of mass spectrometric assays for the yeast proteome. Nat Methods 5(11):913–914
Wenschuh H, Volkmer-Engert R, Schmidt M et al (2000) Coherent membrane supports for parallel microsynthesis and screening of bioactive peptides. Biopolymers 55(3):188–206
Kirkpatrick DS, Gerber SA, Gygi SP (2005) The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modifications. Methods 35(3):265–273
MacLean B, Tomazela DM, Shulman N et al (2010) Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26(7):966–968
Reiter L, Rinner O, Picotti P et al (2011) mProphet: automated data processing and statistical validation for large-scale SRM experiments. Nat Methods 8(5):430–435
Fusaro VA, Mani DR, Mesirov JP, Carr SA (2009) Prediction of high-responding peptides for targeted protein assays by mass spectrometry. Nat Biotechnol 27(2):190–198
Unwin RD, Griffiths JR, Whetton AD (2009) A sensitive mass spectrometric method for hypothesis-driven detection of peptide post-translational modifications: multiple reaction monitoring-initiated detection and sequencing (MIDAS). Nat Protoc 4(6):870–877
Sherwood CA, Eastham A, Lee LW et al (2009) MaRiMba: a software application for spectral library-based MRM transition list assembly. J Proteome Res 8(10):4396–4405
Mead JA, Bianco L, Ottone V et al (2009) MRMaid, the web-based tool for designing multiple reaction monitoring (MRM) transitions. Mol Cell Proteomics 8(4):696–705
Sherman J, McKay MJ, Ashman K, Molloy MP (2009) How specific is my SRM?: the issue of precursor and product ion redundancy. Proteomics 9(5):1120–1123
Mackintosh JA, Veal DA, Karuso P (2005) Fluoroprofile, a fluorescence-based assay for rapid and sensitive quantitation of proteins in solution. Proteomics 5(18):4673–4677
Wisniewski JR, Zougman A, Nagaraj N, Mann M (2009) Universal sample preparation method for proteome analysis. Nat Methods 6(5):359–362
Mallick P, Schirle M, Chen SS et al (2007) Computational prediction of proteotypic peptides for quantitative proteomics. Nat Biotechnol 25(1):125–131
Acknowledgments
This work conducted by the Joint BioEnergy Institute was supported by the Office of Science, Office of Biological and Environmental Research, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.
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Batth, T.S., Keasling, J.D., Petzold, C.J. (2012). Targeted Proteomics for Metabolic Pathway Optimization. In: Keller, N., Turner, G. (eds) Fungal Secondary Metabolism. Methods in Molecular Biology, vol 944. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-122-6_17
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DOI: https://doi.org/10.1007/978-1-62703-122-6_17
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