Direct Amino Acid Analysis Research Articles

Arc-Induced Electrospray Ionization Mass Spectrometry.

Arc-induced electrospray ionization mass spectrometry (AESI-MS) was developed during which alternating current electrospray is simply achieved through the arc plasma. The AESI source exploits the arc's temperature and charge properties to generate aerosols consisting of charged microdroplets. The electrospray region, in which organic molecules are contained within microdroplets, partially overlaps with the arc plasma region. Guided by the electric field, these molecules undergo ionization, yielding ionic target analytes. AESI represents a soft ionization method that combines the mechanisms of atmospheric pressure chemical ionization and electrospray ionization, facilitating the ionization of analytes with wide ranging polarities. The precisely targeted spraying area enhances ion entry into the mass analyzer, thereby enabling excellent ionization efficiency. The AESI source exhibits several notable advantages over the electrospray ionization source, including an elevated but comparable level of active species concentrations and types, simplified mass spectra for direct amino acid analysis, high salt tolerance, versatile analysis of compounds with varying polarities, and reliable quantitative analysis of amino acids in complex matrices. Overall, AESI broadens the methodologies employed to generate microdroplets, providing a technological and scientific framework for creating distinctive electrospray ionization techniques.

Fast, Direct Analysis of Underivatized Amino Acids in Infant Formula

Fast, Direct Analysis of Underivatized Amino Acids in Infant Formula

Host-Guest Self-Assembled Interfacial Nanoarrays for Precise Metabolic Profiling.

Accurate and rapid metabolic profiling of cerebrospinal fluid (CSF) is urgently needed but remains challenging for clinical diagnosis of central nervous system diseases and biomarker discovery. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) holds promise for metabolic analysis. Its low signal reproducibility, however, severely restricts acquisition of quantitative MS data in clinical practice. Herein, a multifunctional self-assembled AuNPs array (MSANA)-based LDI-MS platform for direct amino acids analysis and metabolic profiling in patient CSF samples is developed. MSANA featuring a highly ordered and closely packed two-dimensional nanostructure permits capture and direct analysis of aromatic amino acids by LDI-MS with high selectivity and micromolar sensitivity. Meanwhile, the MSANA-based LDI-MS platform exhibits excellent reproducibility (RSD<10%), largely outperforming the direct matrix spotting approach widely used now (RSD<44%). The platform is successfully used in metabolic profiling of CSF (1µL) within minutes for discrimination of medulloblastoma patients from non-tumor controls. Taken together, the MSANA-based LDI-MS platform shows potential clinical values toward large-scale metabolic diagnostics and pathogenic mechanism study.

Multiple Heart-Cutting Two-Dimensional HPLC-UV Achiral–Chiral Analysis of Branched-Chain Amino Acids in Food Supplements under Environmentally Friendly Conditions

A multiple heart-cutting (mLC-LC) two-dimensional HPLC-UV achiral–chiral method for the direct analysis of branched-chain amino acids (BCAAs) in food supplements under environmentally friendly conditions was developed to cope with the very well-known limited chemoselectivity of chromatographic media for enantioselective analysis. Both achiral and chiral methods were developed in compliance with the main principles of green chromatography. The achiral analysis was performed isocratically with an optimized ion-pair reversed-phase (IP-RP) method based on a water/EtOH (95:5, v/v) mobile phase containing heptafluorobutyric acid (7 mM) as the IP agent. The achiral method was characterized by a very appreciable performance and was validated before the analysis of the real sample. High recovery values for all compounds (from 97% to 101%) were found in the interday evaluation. Additionally, low RSD% values in the long-term period were measured, in the range between 1.1% and 4.8%. Still, an LOQ value of 0.06 mg/mL was established for all compounds. The quantitative analysis of a commercial food supplement revealed that BCAAs were present in amounts very close to those declared by the producer. The enantioselective analysis was carried out through the application of the chiral ligand-exchange chromatography (CLEC) approach, using O-benzyl-(S)-serine ((S)-OBS, 0.5 mM) as the chiral selector and Cu(II) nitrate (0.25 mM) as the metal source in the eluent. Resolution and separation factor values up to 2.31 and 1.43, respectively, were obtained. The two chromatographic systems were connected through a six-port switching valve, and the developed two-dimensional mLC-LC method confirmed the absence of D-enantiomers of BCAAs in the food supplement, as reported in the manufacturer’s label.

3-(4-Carboxybenzoyl)quinoline-2-carboxaldehyde labeling for direct analysis of amino acids in plasma is not suitable for simultaneous quantification of tryptophan, tyrosine, valine, and isoleucine by CE/fluorescence.

Capillary electrophoresis coupled to LED-induced fluorescence detection is a robust and sensitive technique used for amino acids (AA) analysis in biological media, after labeling with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA). We wanted to quantitate in plasma tryptophan (Trp), tyrosine (Tyr), valine (Val), and isoleucine (Ile). Among the different labeled AA-CBQCA, Trp has the lowest fluorescence yield, which makes its detection and quantification very difficult in biological samples such as plasma. We tried to improve Trp analysis by CE/LED-induced fluorescence detection to its maximal sensitivity by using large volume sample stacking as a preconcentration step in our analytical protocol. At pH 9.5, this step caused a drop in resolution during the separation of the four AAs and it was therefore necessary to work at pH 10. We have found that Tyr, Val, Ile, and Trp are detected and well separated from the other AAs, but Trp cannot be quantified in plasma samples, mainly because of the low fluorescence yield of the Trp-CBQCA derivative. The recorded LOD is 0.18μM for Trp-CBQCA in standard solution with a resolution between Trp and Tyr of 1.2, while the LOD is 6μM in plasma with the same resolution. Trp, Tyr, Val, and Ile are, however, efficiently quantified when using a 3 M acetic acid electrolyte and CE associated with capacitively coupled contactless conductivity detection, which also has the advantage of not requiring derivatization or large volume sample stacking. This article demonstrates, for the CE user, that quantitative analysis of these four AA in mouse plasma can be performed by CE-fluorescence after CBQCA labeling, with the exception of Trp. It can be advantageously replaced by CE/capacitively coupled contactless conductivity detection, the only efficient one for Trp, Tyr, Val, and Ile quantification. In this case, the LOD for Trp is 2μM. The four AAs are separated with resolution with neighbors above 1.5.

Direct liquid chromatography tandem mass spectrometry analysis of amino acids in human plasma

Here we describe a new HPLC–MS/MS method using a mixed mode stationary phase and a binary gradient of elution for the rapid separation and quantification of AAs in human plasma without derivatization or ion pairing reagent addition. The sample preparation procedure consists in a single dilution step after protein precipitation with sulfosalicylic acid. The proposed method allows for the unambiguous identification and analysis of 52 AAs and related compounds including the separation of isomers and isobars in an 18 min chromatographic run including the conditioning and the equilibration times. AAs were detected by selective reaction monitoring. Internal calibration was used for the quantification of 37 AAs, including 25 using the corresponding isotopically labeled internal standards. External calibration (no internal standard) was used for five additional analytes. Qualitative detection was achieved for the remaining compounds.Validation studies evaluated accuracy, linearity, within- and between-run precision, lower limits of detection and quantification for 37 amino acids present in commonly used quality control samples. For within-run precision CVs averaged 3.8 % (n = 30) for all compounds. For between-run precision, CVs averaged 8.6 % for all compounds (n = 20). Correlation with the common standard ion-exchange chromatography with post-column derivatization method was also performed for 32 plasma samples. While the proposed method is at least 50 times more sensitive, the data showed good correlation with slopes equal or higher than 0.9 and correlation coefficients mostly higher than 0.90. The method was successfully applied for analysis of plasma samples for detection of inherited disorders of amino acid metabolism.

COMPARATIVE ANALYSIS OF NITROGEN-TO-PROTEIN CONVERSION FACTORS FOR DETERMINING NET PROTEIN CONTENT IN SIX SUPERFOODS

The true protein content of plant foods is best assessed directly by analyzing amino acids quantitatively or indirectly by using a nitrogen-to-protein conversion factor (NPCF) that is computed based on sums of amino acids. The practical way of determining protein in plant foods is by multiplying its total nitrogen (TN) value with a reliable plant-specific NPCF. In this study, three kinds of NPCFs were evaluated for measuring the net-protein content of chia, rye, quinoa, spelt, flax, and hemp. A universal factor of 6.25 was considered NPCF1, while NPCF2s and NPCF3s were based on the sum of amino acids with amide-nitrogen and computed quantities of Asn and Gln using a ratio method, respectively. The TN was minimal in rye (1.34% w/w) and maximal in flax (5.57% w/w). The values of NPCF2 ranged from 5.18 – 5.90 and from 5.45 - 6.22 for NPCF3. The net protein values obtained by NPCF2 were close to the values of direct quantitative analysis of amino acids as compared to other NPCFs. The use of computed Gln and Asn quantities incorrectly increased the amount of available nitrogen in the amino acids, especially in spelt, flax, and hemp; it was about 4% higher than the corresponding TN values, suggesting that the use of the computed method is not appropriate for these plants. NPCF1 overestimated the protein content in all the samples. The degree of overestimation is likely due to the inclusion of non-protein nitrogen in the calculation, which ranged from 5% - 22% in the samples tested.

New Advances in Amino Acid Profiling in Biological Samples by Capillary Electrophoresis-Mass Spectrometry.

Capillary electrophoresis-mass spectrometry (CE-MS) offers a high efficiency microseparation platform for amino acid profiling when analyzing volume-restricted biological samples, such as a dried blood spot punch. Direct analysis of amino acids and their analogs is routinely achieved using strongly acidic buffer conditions under positive-ion mode detection with a coaxial sheath liquid interface for electrospray ionization (ESI). New advances in online sample preconcentration, pre-column chemical derivatization, and/or low flow/sheathless CE-MS interface designs can further improve sensitivity while allowing for resolution of amino acid stereoisomers and labile aminothiols with low nanomolar detection limits. Additionally, multiplexed separations in CE-MS based on serial injection of seven or more samples within a single run greatly boosts sample throughput (<2-3min/sample) without added infrastructure costs while allowing for stringent quality control and signal batch correction. Accurate prediction of the electromigration behavior of amino acids and their analogs offers a convenient approach for structural elucidation that is complementary to high-resolution MS and MS/MS. Simultaneous analysis of amino acids together with other classes of ionic metabolites by CE-MS allows for comprehensive metabolomic screening as required for new advances in clinical medicine, nutritional sciences, and population health.

Direct Analysis of Underivatized Amino Acids in Plant Extracts by LC-MS/MS (Improved Method).

In this chapter we describe a method for quantification of 20 proteinogenic amino acids by liquid chromatography-mass spectrometry which affords neither derivatization nor the use of organic solvents. Analysis of the underivatized amino acids is performed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) in the positive ESI mode. Separation is achieved on a strong cation exchange (SCX) column (Luna 5μ SCX 100Å) with 5% acetic acid in water (A) and 75mM ammonium acetate in water (B). Quantification is accomplished by use of d2-phenylalanine as internal standard achieving limits of detection of 5-50nM. The method was successfully applied for the determination of proteinogenic amino acids in plant extracts.

Accurate and reliable quantification of the protein surface coverage on protein-functionalized nanoparticles

The ability to accurately quantify the protein coverage on nanoparticles is critical for assessing the quality of the surface chemistry and the success of the functionalization process of protein-nanoparticle conjugates. Surface coverage determination is therefore an integral part in the quality control of protein-modified nanoparticles in industrial nanotechnology. In this work, a novel and conventional method was established for direct quantification of the protein surface coverage on metallic nanoparticles. Different concentrations of pepsin were conjugated to gold nanoparticles (GNPs) by a straightforward adsorptive immobilization process as a model system, and a protein quantitation methodology based on the amino acid analysis of the hydrolysate of the protein-GNP conjugates was established. For this purpose, pepsin functionalized GNPs (pepsin-GNP bioconjugates) were processed via in situ hydrolysis with 6N HCl and subsequent derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC reagent). Direct quantitative amino acid analysis was performed based on measuring the intensity of AQC-glycine derivative by high-performance liquid chromatography with fluorescence detection (HPLC-FLD). The method allows for detection of surface coverages as low as 0.1 μg mL−1 pepsin (corresponding to 2.89 × 10−9 mol L−1) in the colloidal solution. Method imprecision for replicated surface coverage determinations was <5% RSD and accuracies, as determined by % recoveries, were always in the 98–118% range. This method allows precise and accurate quantification of protein coverages, even when less than 1% of the protein in the reaction mixture is immobilized. It was found that the degree of surface coverage of adsorptively bound pepsin on GNPs correlated with the pepsin concentrations in the conjugation reaction mixtures. Washing with phosphate buffer removed weakly bound proteins, i.e. the soft protein corona. The adsorption behavior could be described by a Freundlich isotherm model. This direct and reliable method promises great potential for the accurate quantification of protein coverages of various protein-nanoparticle bioconjugates.

Direct analysis of free amino acids by mixed-mode chromatography with tandem mass spectrometry.

We developed a straightforward, robust, and relatively fast method for the analysis of amino acids by mixed-mode high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The method does not involve derivatization and allows the detection of 21 amino acids, representing a wide range of isoelectric points, in less than 40min. Chromatographic separation was governed by a silica-based mixed-mode column providing simultaneous hydrophobic and ion exchange separation mechanisms. The use of tandem mass spectrometry increased selectivity, reducing potential problems associated with poor selectivity in the chromatographic system. For an injection volume of 1μL, we obtained detection limits <3μM for the majority of analytes. For all analytes, a linearity of r > 0.99 was obtained, recovery in matrix was >86%, and the retention times were highly reproducible. The method was successfully applied to soil solution and fungal culture samples, demonstrating the advantages in successfully avoiding issues associated with high amounts of substances that may interfere with derivatization-based methods. This method represents an alternative to derivatization-based methods and can be applied in areas where sample matrices are highly complex.

Direct tandem mass spectrometric analysis of amino acids in plasma using fluorous derivatization and monolithic solid-phase purification

In this study, we developed a novel direct tandem mass spectrometric method for rapid and accurate analysis of amino acids utilizing a fluorous derivatization and purification technique. Amino acids were perfluoroalkylated with 2H,2H,3H,3H-perfluoroundecan-1-al in the presence of 2-picoline borane via reductive amination. The derivatives were purified by perfluoroalkyl-modified silica-based monolithic solid-phase extraction (monolithic F-SPE), and directly analyzed by tandem mass spectrometry using electrospray ionization without liquid chromatographic separation. The perfluoroalkyl derivatives could be sufficiently distinguished from non-fluorous compounds, i.e. the biological matrix, due to their fluorous interaction. Thus, rapid and accurate determination of amino acids was accomplished. The method was validated with human plasma samples and applied to the analysis of amino acids in the plasma of mice with maple syrup urine disease or phenylketonuria.

Direct quantitative analysis of amino acids in fermented beverage of plant extract using high performance liquid chromatography-tandem mass spectrometry

A method was established for underivatized amino acid determination in fermented beverage of plant extract. Samples were diluted with methanol for five times, extracted by ultrasonic vibration for 30 min, and high-speed centrifuged for 15 min at 10,000 r/min. The supernatant was separated and detected by, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The chromatographic column was Venusil ASB C18 (250 mm x 4.6 mm, 5 µm). The elution was performed at a flow rate of 0.5 mL/min using the mobile phases of methanol-acetic acid-water mixture. The MS detector was set as follows: ion source voltage 3 kV, ion source temperature 150 t, solvent temperature 350 t, gas flow rate 800 L/h. The collision gas was argon with a pressure of 0.17 Pa. The quantitation analysis was carried out with peak area in extracted ion chromatograms. Good linearities were acquired in the range of 0.5-200 µmol/L (r2 > 0.99) for the amino acids. The recoveries were between 86% and 110%. There were 16 amino acids in the fermented beverage of plant extract quantitatively analyzed. The method is simple, rapid, accurate and reliable in quantitative analysis of amino acid samples in the fields of pharmaceutical, food and natural products.

Reliable Metabolic Flux Estimation in Escherichia coli Central Carbon Metabolism Using Intracellular Free Amino Acids.

13C metabolic flux analysis (MFA) is a tool of metabolic engineering for investigation of in vivo flux distribution. A direct 13C enrichment analysis of intracellular free amino acids (FAAs) is expected to reduce time for labeling experiments of the MFA. Measurable FAAs should, however, vary among the MFA experiments since the pool sizes of intracellular free metabolites depend on cellular metabolic conditions. In this study, minimal 13C enrichment data of FAAs was investigated to perform the FAAs-based MFA. An examination of a continuous culture of Escherichia coli using 13C-labeled glucose showed that the time required to reach an isotopically steady state for FAAs is rather faster than that for conventional method using proteinogenic amino acids (PAAs). Considering 95% confidence intervals, it was found that the metabolic flux distribution estimated using FAAs has a similar reliability to that of the PAAs-based method. The comparative analysis identified glutamate, aspartate, alanine and phenylalanine as the common amino acids observed in E. coli under different culture conditions. The results of MFA also demonstrated that the 13C enrichment data of the four amino acids is required for a reliable analysis of the flux distribution.

First experience with a fully automated extraction system for simultaneous on‐line direct tandem mass spectrometric analysis of amino acids and (acyl‐)carnitines in a newborn screening setting

For Newborn Screening (NBS) programs all over the world whole blood dried on filter paper, also referred to as dried blood spots (DBS), has been the standard specimen for decades. In recent years DBS have attracted the attention of pharmaceutical companies, mostly due to the low volume of collected sample and simplified, therefore more cost-efficient, transportation requirements. However, the classical NBS workflow did not totally fulfil the needs of their studies, especially with respect to high-throughput unassisted sample processing for tandem mass spectrometric (MS/MS) analysis. Automated on-line extraction systems for direct analysis have already been tested and proved to be suitable for these pharmaceutical applications. The suitability of the automated CAMAG DBS-MS 500 interface for simultaneous detection of amino acids and (acyl-)carnitines has been tested together with an Acquity TQD tandem mass spectrometer from Waters and MassChrom stable isotope labelled internal standards from Chromsystems. No chromatographic sample treatment was applied; instead, the extract was directly injected into the MS/MS instrument. The feasibility of the instrumental setting for the routine newborn screening was tested on original samples coming from previously diagnosed patients. The performance of the automated extraction technique and its application in preliminary quantitative screening for amino acids and (acyl-)carnitines for NBS showed very promising results. Several samples from patients, each diagnosed with one of four different inborn errors of metabolism (IEM), were tested and the correlation with the conventional punch-and-elute approach was very good. Although the presented method still needs further optimization, our study clearly shows the possibility to use direct on-line analysis in the NBS setting. Our report on direct on-line analysis of newborn samples is a first approach in the development of a fully automated screening method for NBS analysis. With regard to the chemical properties of the analytes, the study resulted in a readily applicable screening method.

Direct quantitative analysis of aromatic amino acids in human plasma by four-way calibration using intrinsic fluorescence: Exploration of third-order advantages

A novel intrinsic fluorescence method for the direct determination of l-phenylalanine, l-tyrosine, and l-tryptophan in human plasma is presented. By using fluorescence excitation-emission-pH-sample data array in combination with four-way calibration method based on the quadrilinear component model, the proposed approach successfully achieved quantitative analysis of the aromatic amino acids in human plasma, even in the presence of an unknown, uncalibrated serious interferent. It needs little preparation, uses the "mathematical separation" instead of "analytical separation", what makes it fast and environmentally friendly. Satisfactory results have been achieved for calibration set, validation set, and prediction set. The ranges for phenylalanine, tyrosine, and tryptophan are 2.0 × 10(3)-20.0 × 10(3), 50.0-500.0, and 20.0-200.0 ng mL(-1) respectively. Average spike recoveries (mean ± standard deviation) are 93.3 ± 7.7%, 104.3 ± 6.6%, and 99.5 ± 9.0% respectively. The real concentrations in human plasma are 10.2 ± 0.3, 6.6 ± 0.1, and 5.3 ± 0.1 μg mL(-1) respectively, which are consistent with the results obtained by LC-MS/MS method and reference values. In addition, we explored the third-order advantages through the real four-way array; it has shown that higher resolving power is one of the main advantages of higher-order tensor calibration method. These results demonstrated that the proposed method is sensitive, accurate, and efficient for direct quantitative analysis of aromatic amino acids in human plasma.

Detection of tropomyosin and determination of proteins in crustacean oils

Tropomyosin is known to be the main allergen in crustaceans and the objective of this study was to investigate if this protein could be detected in commercial crustacean oils from Antarctic krill (Euphausia superba) and the zooplankton Calanus finmarchicus. We also examined the possibility of determining the protein content in the oils by direct amino acid analysis. Western blotting showed that a commercial antibody against shrimp tropomyosin cross-reacted with a protein of similar size in Antarctic krill and C. finmarchicus. The protein tentatively identified as tropomyosin, was also detected in krill oil products, but not in oils from C. finmarchicus. The acetone–heptane method used for extracting proteins in the oils is however not optimal. Other extraction methods should therefore be considered when investigating the presence of allergenic proteins in oils. Direct amino acid analysis on oils should be further explored as a method for determining the total amount of proteins present.

Polyfunctional low-capacity cation-exchange packing material for the separation of underivatized amino acids

A polyfunctional low-capacity cation-exchange column-packing material was developed for the simultaneous separation and determination of underivatized 20 amino acids including 16 proteinogenic ones using a binary gradient HPLC. The new packing material was prepared by sulfoacylating a highly crosslinked macroreticular poly(styrene-divinylbenzene) copolymer of 3μm in diameter commercially available for size-exclusion chromatography. The polyfunctionality could derive from unintentional carboxy groups innately present in the base polymers probably came from polymerization initiators in addition to the intentionally introduced sulfopropionyl groups, which was certified and evaluated by measuring dynamic capacity curves of the cation exchanger. Sixteen underivatized proteinogenic amino acids were separated in 23min using a binary high-pressure gradient elution system with two liquids of 1mmolL−1 H3PO4 and 20mmolL−1 NaH2PO4/30% (v/v) CH3CN, which led to the cycle time of 25min. In the case of separation of 20 amino acids, the cycle time was 27min. The developed low-capacity cation-exchange chromatography with post-column fluorescence detection was sufficiently quantitative, providing linear calibration lines ranged through almost three digit for all analytes. The detection limits were calculated as nmolL−1 order of magnitude with 20-μL injection. The method was applicable to the direct analysis of urinary amino acids of diagnostic markers for inborn errors of metabolism.

An UPLC-ESI-MS/MS Assay Using 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate Derivatization for Targeted Amino Acid Analysis: Application to Screening of Arabidopsis thaliana Mutants

In spite of the large arsenal of methodologies developed for amino acid assessment in complex matrices, their implementation in metabolomics studies involving wide-ranging mutant screening is hampered by their lack of high-throughput, sensitivity, reproducibility, and/or wide dynamic range. In response to the challenge of developing amino acid analysis methods that satisfy the criteria required for metabolomic studies, improved reverse-phase high-performance liquid chromatography-mass spectrometry (RPHPLC-MS) methods have been recently reported for large-scale screening of metabolic phenotypes. However, these methods focus on the direct analysis of underivatized amino acids and, therefore, problems associated with insufficient retention and resolution are observed due to the hydrophilic nature of amino acids. It is well known that derivatization methods render amino acids more amenable for reverse phase chromatographic analysis by introducing highly-hydrophobic tags in their carboxylic acid or amino functional group. Therefore, an analytical platform that combines the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) pre-column derivatization method with ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) is presented in this article. For numerous reasons typical amino acid derivatization methods would be inadequate for large scale metabolic projects. However, AQC derivatization is a simple, rapid and reproducible way of obtaining stable amino acid adducts amenable for UPLC-ESI-MS/MS and the applicability of the method for high-throughput metabolomic analysis in Arabidopsis thaliana is demonstrated in this study. Overall, the major advantages offered by this amino acid analysis method include high-throughput, enhanced sensitivity and selectivity; characteristics that showcase its utility for the rapid screening of the preselected plant metabolites without compromising the quality of the metabolic data. The presented method enabled thirty-eight metabolites (proteinogenic amino acids and related compounds) to be analyzed within 10 min with detection limits down to 1.02 × 10−11 M (i.e., atomole level on column), which represents an improved sensitivity of 1 to 5 orders of magnitude compared to existing methods. Our UPLC-ESI-MS/MS method is one of the seven analytical platforms used by the Arabidopsis Metabolomics Consortium. The amino acid dataset obtained by analysis of Arabidopsis T-DNA mutant stocks with our platform is captured and open to the public in the web portal PlantMetabolomics.org. The analytical platform herein described could find important applications in other studies where the rapid, high-throughput and sensitive assessment of low abundance amino acids in complex biosamples is necessary.

Dynamic Monitoring of Newly Synthesized Proteomes: Up-Regulation of Myristoylated Protein Kinase A During Butyric Acid Induced Apoptosis

Post-translational modification (PTM) is a highly dynamic yet precisely controlled process by which most eukaryotic proteins are chemically diversified. Many critical cellular responses are mediated through PTMs, which lead to modulation of enzyme activity, protein conformation, protein–protein interaction, and cellular localization. Analysis of these modifications at the proteome level could provide invaluable biological insight but remains a technically challenging undertaking. Traditionally, PTMs have been studied by standard molecular biology techniques involving tedious isolation of individual proteins and subsequent direct detection and analysis of amino acids bearing the modification. Recent advances in mass spectrometry, when combined with stable-isotope or metabolic labeling approaches, have enabled several large-scale studies of PTMs and their dynamics. These methods, however, analyze PTM changes of all proteins (old and new) present in the cell at the time of sampling and thus are only able to evaluate PTM dynamics at the ensemble level. With unnatural metabolic building blocks and in vivo compatible conjugation chemistries becoming increasingly available (Scheme 1), we sought to develop a proteomic strategy for the detection and identification of newly synthesized proteomes and their PTMs (Figure 1). We envisioned several advantages of studying the PTM dynamics of newly synthesized proteomes: 1) this method decreases the complexity of the proteome and enables the identification of PTM changes that occur in a predefined protein synthesis window; 2) it gives an accurate estimate of the time scale of different PTM events in transforming newly synthesized, modification-free proteins into mature functional entities; 3) it permits PTM analysis of primary protein synthesis responses to internal and external cues. To isolate a newly synthesized proteome, wemade use of BONCAT (bio-orthogonal noncanonical amino acid tagging), which uses the known methionine surrogates azidohomoalanine (AHA) and homopropargylglycine (HPG) for metabolic incorporation into newly synthesized proteins (Scheme 1, blue) and the corresponding alkyneor azide-modified biotin reporter (Scheme 1, bottom) for subsequent proteome isolation. To monitor dynamic changes of an PTM event, we fed growing cells with an azideor alkynecontaining sugar, fatty acid, or lipid building block (Scheme 1, orange). It should be noted that while our work was in progress, Hang and co-workers reported a tandem labeling and detection method to monitor the dynamic acylation of LCK (a tyrosine kinase) and its turnover. Their work focused on the study of single PTM events (i.e. protein palmitoylation) of a specific protein (i.e., LCK) in a proteome. The work herein, while conceptually similar, greatly expands the scope of this double metabolic incorporation strategy by successfully demonstrating, for the first Scheme 1. Methionine surrogates (blue) and unnatural metabolite PTM probes (orange) form bio-orthogonal pairs for compatible double metabolic incorporation. Those forming pairs are boxed in the same group. Azideand alkyne-containing fluorophore and biotin reporters are also shown (bottom).