Development of microwave-assisted acid hydrolysis of proteins using a commercial microwave reactor and its combination with LC–MS for protein full-sequence analysis

Chen, Lu; Wang, Nan; Li, Liang

doi:10.1016/j.talanta.2014.05.042

Cited by 9 publications

(4 citation statements)

References 32 publications

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“…MS emerged as a powerful diagnostic tool in the identification of Hb variants in the last three decades [ 10 , 11 , 12 ]. Two different approaches—top−down [ 30 , 31 ] and bottom−up, were generally used to characterize and identify Hb variants by MS [ 32 ]. In the top−down proteomics approach, intact protein samples are subjected to fragmentation and tandem MS analysis is performed for acquiring protein sequence coverage, whereas in the bottom−up proteomics approach, proteins are digested with enzymes to form peptides and then these peptides are further subjected to MS and tandem MS for obtaining protein sequence coverage.…”

Section: Discussionmentioning

confidence: 99%

Optimization and Identification of Single Mutation in Hemoglobin Variants with 2,2,2 Trifluoroethanol Modified Digestion Method and Nano−LC Coupled MALDI MS/MS

et al. 2022

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Hemoglobin (Hb) variants arise due to point mutations in globin chains and their pathological treatments rely heavily on the identification of the nature and location of the mutation in the globin chains. Traditional methods for diagnosis such as HPLC and electrophoresis have their own limitations. Therefore, the present study aims to develop and optimize a specific method of sample processing that could lead to improved sequence coverage and analysis of Hb variants by nano LC−MALDI MS/MS. Methods: In our study, we primarily standardized various sample processing methods such as conventional digestion with trypsin followed by 10% acetonitrile treatment, digestion with multiple proteases like trypsin, Glu−C, Lys−C, and trypsin digestion subsequent to 2,2,2 trifluoroethanol (TFE) treatment. Finally, the peptides were identified by LC−MALDI MS/MS. All of these sample processing steps were primarily tested with recombinant Hb samples. After initial optimization, we found that the TFE method was the most suitable one and the efficiency of this method was applied in Hb variant identification based on high sequence coverage. Results: We developed and optimized a method using an organic solvent TFE and heat denaturation prior to digestion, resulting in 100% sequence coverage in the β−chains and 95% sequence coverage in the α−chains, which further helped in the identification of Hb mutations. A Hb variant protein sequence database was created to specify the search and reduce the search time. Conclusion: All of the mutations were identified using a bottom−up non−target approach. Therefore, a sensitive, robust and reproducible method was developed to identify single substitution mutations in the Hb variants from the sequence of the entire globin chains. Biological Significance: Over 330,000 infants are born annually with hemoglobinopathies and it is the major cause of morbidity and mortality in early childhood. Hb variants generally arise due to point mutation in the globin chains. There is high sequence homology between normal Hb and Hb variant chains. Due to this high homology between the two forms, identification of variants by mass spectrometry is very difficult and requires the full sequence coverage of α− and β−chains. As such, there is a need for a suitable method that provides 100% sequence coverage of globin chains for variant analysis by mass spectrometry. Our study provides a simple, robust, and reproducible method that is suitable for LC−MALDI and provides nearly complete sequence coverage in the globin chains. This method may be used in the near future in routine diagnosis for Hb variant analysis.

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Section: Discussionmentioning

confidence: 99%

Optimization and Identification of Single Mutation in Hemoglobin Variants with 2,2,2 Trifluoroethanol Modified Digestion Method and Nano−LC Coupled MALDI MS/MS

et al. 2022

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“…The traditional acid hydrolyses method generally performed with 6N HCl at 110°C for 24 hrs (Damm et al 2010;Afiuni-Zadeh et al 2011). The microwave assisted hydrolysed method to develop recently as a useful tool for a range of protein sample preparation for proteome analysis (Chen et al 2014;Wang and Li 2010). This method helps to reduce hydrolysing time significantly as a major advantage.…”

Section: Introductionmentioning

confidence: 99%

Validation of microwave-assisted sample extraction-high performance liquid chromatography (HPLC) method for quantitative analysis of hydrolysed amino acids in aquatic biological samples

Jayasinghe¹,

Jinadasa²

2019

Sri Lanka J. Aquat.

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A sensitive and widely applicable High-Performance Liquid Chromatography (HPLC) with fluorescence detector (FLD) method for determination hydrolysed amino acids in fish and aquatic products was developed and validated through study. The method involved a microwave-assisted sample extraction and protein hydrolysed with the hydrochloric acid and propionic acid. The separation was performed by Shim-pack HR-ODS column with a gradient mobile phase. The total separation time was 25 min per run. The method showed satisfactory linearity, repeatability, accuracy, and limit of detection for 21 amino acids with the 1-100 mg/L calibration range based on 10 µL injection. The fish, fish feed, canned fish and seaweeds samples were analysed successfully by using this developed method.

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“…13 Microwave "intensication" is also a common tool for proteomics. [14][15][16] In the microwave technique the sample can be hydrolyzed in liquid or gas phase mode. Microwave-assisted vapor phase acid hydrolysis is a relatively newer technique (acid vapors are used in the process instead of liquid acid).…”

Section: Introductionmentioning

confidence: 99%

Determination of underivatized amino acids in microsamples of a yeast nutritional supplement by LC-MS following microwave assisted acid hydrolysis

2016

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Development of microwave-assisted acid hydrolysis of proteins using a commercial microwave reactor and its combination with LC–MS for protein full-sequence analysis

Cited by 9 publications

References 32 publications

Optimization and Identification of Single Mutation in Hemoglobin Variants with 2,2,2 Trifluoroethanol Modified Digestion Method and Nano−LC Coupled MALDI MS/MS

Optimization and Identification of Single Mutation in Hemoglobin Variants with 2,2,2 Trifluoroethanol Modified Digestion Method and Nano−LC Coupled MALDI MS/MS

Validation of microwave-assisted sample extraction-high performance liquid chromatography (HPLC) method for quantitative analysis of hydrolysed amino acids in aquatic biological samples

Determination of underivatized amino acids in microsamples of a yeast nutritional supplement by LC-MS following microwave assisted acid hydrolysis

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