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

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          Abstract

          Background

          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, avoiding 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 hinder precise protein quantitation using AAA.

          Results

          In this study, a hydrophilic interaction chromatography based method was used to separate all proteinogenic amino acids, including isobaric compounds leucine and isoleucine, prior to detection by multiple reaction monitoring with LC–MS/MS. Through inclusion of commercially available isotopically labeled ( 13C, 15N) amino acids as internal standards we adapted an isotopic dilution strategy for amino acid-based quantification of proteins. Three hydrolysis methods were tested with ubiquitin, bovine serum albumin, (BSA), and a soy protein biological reference material (SRM 3234; NIST) resulting in protein estimates that were 86–103%, 82–94%, and 90–99% accurate for the three protein samples respectively. The methane sulfonic acid hydrolysis approach provided the best recovery of labile amino acids including: cysteine, methionine and tryptophan that are challenging to accurately quantify.

          Conclusions

          Accurate determination of protein quantity and amino acid composition in heterogeneous biological samples is non-trivial. Recent advances in chromatographic phases and LC–MS/MS based methods, along with the availability of isotopic standards can minimize difficulties in analysis and improve protein quantitation. A robust method is described for high-throughput protein quantification and amino acid compositional analysis. Since accurate measurement of protein quality and quantity are a requirement for many biological studies that relate to crop improvement or more generally, our understanding of metabolism in living systems, we envision this method will have broad applicability.

          Electronic supplementary material

          The online version of this article (10.1186/s13007-019-0430-z) contains supplementary material, which is available to authorized users.

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          Most cited references55

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          Automatic Recording Apparatus for Use in Chromatography of Amino Acids

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            Converting nitrogen into protein--beyond 6.25 and Jones' factors.

            The protein content in foodstuffs is estimated by multiplying the determined nitrogen content by a nitrogen-to-protein conversion factor. Jones' factors for a series of foodstuffs, including 6.25 as the standard, default conversion factor, have now been used for 75 years. This review provides a brief history of these factors and their underlying paradigm, with an insight into what is meant by "protein." We also review other compelling data on specific conversion factors which may have been overlooked. On the one hand, when 6.25 is used irrespective of the foodstuff, "protein" is simply nitrogen expressed using a different unit and says little about protein (s.s.). On the other hand, conversion factors specific to foodstuffs, such as those provided by Jones, are scientifically flawed. However, the nitrogen:protein ratio does vary according to the foodstuff considered. Therefore, from a scientific point of view, it would be reasonable not to apply current specific factors any longer, but they have continued to be used because scientists fear opening the Pandora's box. But because conversion factors are critical to enabling the simple conversion of determined nitrogen values into protein values and thus accurately evaluating the quantity and the quality of protein in foodstuffs, we propose a set of specific conversion factors for different foodstuffs, together with a default conversion factor (5.6). This would be far more accurate and scientifically sound, and preferable when specifically expressing nitrogen as protein. These factors are of particular importance when "protein" basically means "amino acids," this being the principal nutritional viewpoint.
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              Hydrophilic interaction liquid chromatography (HILIC)—a powerful separation technique

              Hydrophilic interaction liquid chromatography (HILIC) provides an alternative approach to effectively separate small polar compounds on polar stationary phases. The purpose of this work was to review the options for the characterization of HILIC stationary phases and their applications for separations of polar compounds in complex matrices. The characteristics of the hydrophilic stationary phase may affect and in some cases limit the choices of mobile phase composition, ion strength or buffer pH value available, since mechanisms other than hydrophilic partitioning could potentially occur. Enhancing our understanding of retention behavior in HILIC increases the scope of possible applications of liquid chromatography. One interesting option may also be to use HILIC in orthogonal and/or two-dimensional separations. Bioapplications of HILIC systems are also presented. Figure  
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                Author and article information

                Contributors
                skambhampati@danforthcenter.org
                jli@danforthcenter.org
                Bevans@danforthcenter.org
                Doug.Allen@ars.usda.gov
                Journal
                Plant Methods
                Plant Methods
                Plant Methods
                BioMed Central (London )
                1746-4811
                11 May 2019
                11 May 2019
                2019
                : 15
                : 46
                Affiliations
                [1 ]ISNI 0000 0004 0466 6352, GRID grid.34424.35, Donald Danforth Plant Science Center, ; St. Louis, MO USA
                [2 ]ISNI 0000 0004 0404 0958, GRID grid.463419.d, United States Department of Agriculture, Agricultural Research Service, ; St. Louis, MO USA
                Author information
                http://orcid.org/0000-0001-8599-8946
                Article
                430
                10.1186/s13007-019-0430-z
                6511150
                31110556
                5ebb8be4-6ed5-489a-bf68-97811b83802d
                © The Author(s) 2019

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 12 February 2019
                : 25 April 2019
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100012009, United Soybean Board;
                Award ID: 1820-152-0134
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100008982, National Science Foundation;
                Award ID: 1616820
                Award ID: 1427621
                Award Recipient :
                Funded by: USDA National Institute of Food and Agriculture
                Award ID: 2017-67013-26156
                Award Recipient :
                Categories
                Methodology
                Custom metadata
                © The Author(s) 2019

                Plant science & Botany
                hilic,chromatography,isobaric compounds,amino acids,protein hydrolysis,protein quantification,lc–ms/ms,soybeans,isotopes

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