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

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 ( 13 C, 15 N) 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.

Chromatographic Methods for Evaluation of the Amino Acid and Fatty Acid Compositions Related with Pork Quality

“…The authors established the pH influence of the mobile phase on the separation factors of amino acids, proposed to optimize the conditions for acid hydrolysis of samples by performing a sample preparation procedure, including acid hydrolysis of samples and modification of amino acids with a solution of phenyl isothiocyanate. A hydrolysis and other technique have been described previously and the resulting AA solutions were subjected to chromatographic analysis [13][14][15][31][32][33].…”

Physico-chemical characterization and transcriptome analysis of 5-methyltryptophan resistant lines in rice

“…Analysis did not employ acid hydrolysis condition to avoid loss of Trp known to occur in most acidic solutions. A similar detailed protocol can be adapted for HPLC–MS/MS instrumentation, chromatographic and MS parameters [21].…”

On the Inverse Correlation of Protein and Oil: Examining the Effects of Altered Central Carbon Metabolism on Seed Composition Using Soybean Fast Neutron Mutants

“…Protein quantification was performed by hydrolyzing the peptide bonds and quantifying the resulting individual amino acids as described in Kambhampati et al [45]. Protein hydrolysis was performed in 100 µL of 4 M methanesulfonic acid with 0.2% tryptamine spiked with 20 µL of 1 mM 13 C 15 N-labeled amino acid standard mix.…”

“…Samples were then completely dried down and 1 mL 50% MeOH was added to resuspend hydrolyzed amino acids prior to filtering with a 0.8 µM PES membrane centrifuge filter. Chromatography and mass spectrometry conditions for separation and detection of amino acids were identical to the method described in Kambhampati et al [45].…”