Total Protein Analysis in Algae via Bulk Amino Acid Detection: Optimization of Amino Acid Derivatization after Hydrolysis with <i>O</i>-Phthalaldehyde 3-Mercaptopropionic Acid (OPA-3MPA)

Cuchiaro, Jamie; Laurens, Lieve M.L.

doi:10.1021/acs.jafc.9b00884

Cited by 18 publications

(6 citation statements)

References 37 publications

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“…Based on the HRMS data of 451 endogenous primary amine metabolites, the possible molecular formulas can be calculated. According to the reaction mechanism (shown in Supporting Information, Figure S1, including PH and reaction mechanism), in the presence of the reducing agent 3-MPA, OPA, and primary amines might produce OPA-3-MPA-primary amine complex products under alkaline conditions. ,, Since these products exist in the form of [OPA-3-MPA-primary amine + H] + , in the identification process of Thermo Xcalibur 3.0.63 software, the basic element composition of [OPA-3-MPA-primary amine + H] + could be determined and include at least 1 N, 11 C, 11 H, 2 O, and 1 S. After deducting the increased molecular weight after derivatization and considering the molecular weight of possible primary amines, the structure was determined by matching the molecular formula with the HMDB and Metlin database according to the secondary mass spectral information. For the compounds with multiple isomers, the nonprimary amines (in which case OPA derivatization might not occur) were excluded first before expected compound exclusion.…”

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…In this study, O-phthalaldehyde (OPA) and O-phthalaldehyde-D 4 (OPA-D 4 ) labels were selected to conduct metabolomics research on primary amine compounds in human serum samples. 30 The comparison of OPA derivatization reagents with other derivatization reagents is provided as Supporting Information. One of the interesting findings is that the accurate mass number difference between H and its stable isotope D is 1.00627 Da, and the difference in mass defects between the two is approximately 6 mDa, which contributes to the mass number difference of 4.02511 Da and mass defect difference of 25.11 mDa between OPA and OPA-D 4 .…”

Section: ■ Introductionmentioning

confidence: 99%

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MS-IDF: A Software Tool for Nontargeted Identification of Endogenous Metabolites after Chemical Isotope Labeling Based on a Narrow Mass Defect Filter

et al. 2022

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Chemical isotope labeling liquid chromatography mass spectrometry (LC-MS) is an emerging metabolomic strategy for the quantification and characterization of small molecular compounds in biological samples. However, its subsequent data analysis is not straightforward due to a large amount of data produced and interference of biological matrices. In order to improve the efficiency of searching and identification of target endogenous metabolites, a new software tool for nontargeted metabolomics data processing called MS-IDF was developed based on the principle of a narrow mass defect filter. The developed tool provided two function modules, including IsoFinder and MDFinder. The IsoFinder function module applied a conventional peak extraction method by using a fixed mass differences between the heavy and light labels and by the alignment of chromatographic retention time (RT). On the other hand, MDFinder was designed to incorporate the accurate mass defect differences between or among stable isotopes in the peak extraction process. By setting an appropriate filter interval, the target metabolites can be efficiently screened out while eliminating interference. Notably, the present results showed that the efficiency in compound identification using the new MDFinder module was nearly doubled as compared to the conventional IsoFinder method (an increase from 259 to 423 compounds). The Matlab codes of the developed MS-IDF software are available from github at https://github.com/jydong2018/ MS_IDF. Based on the MS-IDF software tool, a novel and effective approach from nontargeted to targeted metabolomics research was developed and applied to the exploration of potential primary amine biomarkers in patients with schizophrenia. With this approach, potential biomarkers, including N,N-dimethylglycine, S-adenosine-L-methionine, DL-homocysteine, and spermidine, were discovered.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

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MS-IDF: A Software Tool for Nontargeted Identification of Endogenous Metabolites after Chemical Isotope Labeling Based on a Narrow Mass Defect Filter

et al. 2022

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“…Each sample was digested in 6N HCl for 24 h until hydrolysis. Total amino nitrogen was determined using the nitrogen O-phthaldialdehyde (NOPA) procedure ( Cuchiaro and Laurens, 2019 ), and the protein content in the samples by applying an amino-nitrogen-to-protein conversion factor of 6.25 ( Soto-Sierra et al, 2021 ).…”

Section: Methodsmentioning

confidence: 99%

Feasibility of membrane ultrafiltration as a single-step clarification and fractionation of microalgal protein hydrolysates

Sierra

Nikolov

2022

Front. Bioeng. Biotechnol.

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Protein hydrolysates are one of the most valuable products that can be obtained from lipid-extracted microalgae (LEA). The advantages of protein hydrolysates over other protein products encompass enhanced solubility, digestibility, and potential bioactivity. The development of an economically feasible process to produce protein hydrolysates depends on maximizing the recovery of hydrolyzed native protein from the lipid-extracted algal biomass and subsequent fractionation of hydrolyzed protein slurry. Previously, we reported a method for fractionation of enzymatically generated protein hydrolysates by acidic precipitation of algal cell debris and unhydrolyzed protein, precipitate wash, centrifugation, and depth filtration. The present study evaluates tangential flow ultrafiltration as a single-step alternative to centrifugation, precipitate wash, and depth filtration. The results demonstrate that the tangential flow ultrafiltration process has a potential that deserves further investigation. First, the membrane diafiltration process uses a single and easily scalable unit operation (tangential flow filtration) to separate and “wash out” hydrolyzed protein from the algal residue. Second, the protein recovery yield achieved with the tangential flow process was >70% compared to 64% previously achieved by centrifugation and depth filtration methods. Finally, protein hydrolysates obtained by membrane ultrafiltration exhibited slightly better heat and pH stability.

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“…Protein was determined using the Lowry method assay (Noble and Bailey, 2009) at 660 nm using gelatine as standard. Total peptides were analysed in the supernatant samples as described by Cuchiaro and Laurens (2019). VFAs were quantified on a Trace gas chromatograph equipped with a Thermo TR-WAX column (30 m x ID 0.32 mm x thickness of 0.25 µm) connected to a FID detector as described by Sudmalis et al (2018).…”

Section: Analysesmentioning

confidence: 99%

Volatile fatty acids and methane production from protein

Duong¹

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Many industrial wastes and wastewaters, e.g. dairy, beverage, slaughterhouse wastewater, and food processing waste streams contain appreciable quantities of protein. Anaerobic treatment is appropriate for wastewaters and wastes rich in organics to achieve pollution control and resource recovery, e.g. energy rich methane or important chemical platforms such as volatile fatty acids (VFA) or added-valuable amino acids. However, the understanding of the hydrolysis, especially of proteins, is still limited, resulting in non-optimized anaerobic systems dealing with these protein rich wastewaters. Therefore, this thesis aimed to investigate anaerobic conversion of proteins, focussing on the hydrolysis process to accomplish sufficient protein degradation for VFA and methane production. Batch and completely-stirred tank reactor (CSTR) experiments were carried out to study the effect of different environmental and microbial factors on hydrolysis and further degradation of gelatine, a model protein, at mesophilic conditions. Results show that in contrast to earlier suggestions in literature, carbohydrates did not directly affect the protein hydrolysis rates either under methanogenic or non-methanogenic conditions at neutral pH. However, the high VFA concentrations strongly inhibited the protein hydrolysis rate in the batch experiments. Methanogenesis did not stimulate the rate of hydrolysis and acidification of protein at pH 7. Yet, protein hydrolysis was inhibited at pH 5. The hydrolysis rate constant for protein at pH 5 (0.05 L g -1 VSS day -1 ) was much lower than at pH 7 (0.62 L g -1 VSS day -1 ). Even long-term exposure (480 days) of the microbial population to pH 5 did not result in an enhanced hydrolysis of dissolved protein. Hydrolysis always is the rate-limiting step of protein degradation at pH 5 between a solid retention time (SRT) of 12 and 30 days. At pH 7, protein degradation was limited by hydrolysis at SRTs >8 days or acidogenesis at SRTs ≤ 8 days. The pH also determined the VFA product spectra.Different mathematical models for kinetics of hydrolysis were tested for the steady states of the CSTR pH 7 during 600 days. The modelling results indicated that the high concentrations of amino acids may inhibit hydrolysis of proteins. The findings in this study give direction how to solve problems associated with insufficient protein degradation and how to design and operate anaerobic treatment processes for protein rich wastewaters. For methane production, the reactor design should be based on methanogenesis being the slowest process if the wastewater is mainly comprised of dissolved proteins. Regarding the VFA production, the optimum volumetric VFA productivity was 2.3 g CODVFA L -1 day -1 at SRT 10 days and pH 7. Recommendations for improving the VFA productivity include design of anaerobic granular-based reactors to attain high biomass concentrations, operation at an optimum pH between pH 5 and pH 7 to avoid methanogenesis and active separation of VFA to avoid the inhibitory effect of the VFA on the protein hydr...

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Total Protein Analysis in Algae via Bulk Amino Acid Detection: Optimization of Amino Acid Derivatization after Hydrolysis with O-Phthalaldehyde 3-Mercaptopropionic Acid (OPA-3MPA)

Cited by 18 publications

References 37 publications

MS-IDF: A Software Tool for Nontargeted Identification of Endogenous Metabolites after Chemical Isotope Labeling Based on a Narrow Mass Defect Filter

MS-IDF: A Software Tool for Nontargeted Identification of Endogenous Metabolites after Chemical Isotope Labeling Based on a Narrow Mass Defect Filter

Feasibility of membrane ultrafiltration as a single-step clarification and fractionation of microalgal protein hydrolysates

Volatile fatty acids and methane production from protein

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