Accurate and efficient amino acid analysis for protein quantification using hydrophilic interaction chromatography coupled tandem mass spectrometry

Authors: KAMBHAMPATI, SHRIKAAR - Danforth Plant Science Center; LI, JIA - Danforth Plant Science Center; EVANS, BRADLEY - Danforth Plant Science Center; Allen, Douglas - Doug

Submitted to: Plant Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/25/2019
Publication Date: 5/11/2019
Publication URL: http://handle.nal.usda.gov/10113/6487969
Citation: Kambhampati, S., Li, J., Evans, B.S., Allen, D.K. 2019. Accurate and efficient amino acid analysis for protein quantification using hydrophilic interaction chromatography coupled tandem mass spectrometry. Plant Methods. 15:46. https://doi.org/10.1186/s13007-019-0430-z.
DOI: https://doi.org/10.1186/s13007-019-0430-z

Interpretive Summary: Proteins are one of the essential macromolecules to life. In plants they fulfill enzymatic, signaling, structural and storage biological functions, are crucial to human and animal nutrition, and can serve to meet other renewable resource, and pharmaceutical needs. While proteins are ubiquitous, quantifying protein levels in biomass is challenging. There are many methods; some reduce biomass to elemental forms and therefore may overestimate protein due to contamination of elements from other compounds, others rely on spectrophotometric assays and are limited by the ability to solubilize protein using buffers that are compatible with the assay procedure. We describe the development of a method that is quantitative and can account for all of the protein within standards and biological matrices. The method is based on hydrolyzing protein then separating its amino acid components and quantifying them individually using a liquid chromatography and mass spectrometry-based approach. Through the use of isotopically labeled amino acids and standards we demonstrate the ability to quantify protein accurately. Accurate quantification is important to efforts in crop breeding that are predicated on enhancing nutrition by changing protein levels and composition and to developing renewable alternative feed stocks for industry.

Technical Abstract: Methods used to quantify protein from biological samples are often inaccurate with significant variability that requires care to minimize. The errors result from losses during protein preparation and purification and false detection of interfering compounds or elements. Amino acid analysis (AAA) involves a series of chromatographic techniques that can be used to measure protein levels, subverting some difficulties and providing specific compositional information. However, unstable derivatives, that are toxic and can be costly, incomplete reactions, inadequate chromatographic separations, and the lack of a single hydrolysis method with sufficient recovery of all amino acids prevent precise protein quantitation using AAA. In this study, a hydrophilic interaction chromatography (HILIC) based method was developed to separate all proteinogenic amino acids and detect them with multiple reaction monitoring using LC-MS/MS. Through inclusion of commercially available (13C, 15N) amino acids as internal standards, the isotopic dilution method for AAA accounts for differential recoveries including low amounts of labile amino acids, (cysteine, methionine and tryptophan). In addition, MS detection is not retention time dependent or impacted by contaminating chromophores and has enhanced sensitivity and accuracy over a broad dynamic range. Three hydrolysis methods were tested with ubiquitin, bovine serum albumin and a soybean protein reference resulting in protein estimates that were 86-103%, 82-94%, and 90-99%, accurate respectively. Quantitative measurements of proteins and amino acids are broadly important for crop improvement for nutrition or renewable feed stock generation, and also efforts in medicine and human health; including both fundamental and applied studies in science.