Peptic and tryptic digestion of peptides and proteins monitored by capillary electrophoresis with contactless conductivity detection

Schuchert‐Shi, Aiping; Hauser, Peter C.

doi:10.1016/j.ab.2009.01.025

Cited by 36 publications

(19 citation statements)

References 41 publications

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“…LODs are even higher for neutral species that require indirect detection. The feasibility of monitoring the peptic and tryptic digestion of peptides and proteins with EMMA‐C 4 D has been demonstrated by Schuchert‐Shi and Hauser 65. Recently, this group utilized C 4 D to study acetylcholinesterase inhibitors on‐line 66.…”

Section: In‐capillary Assaymentioning

confidence: 99%

Recent developments and applications of EMMA in enzymatic and derivatization reactions

2011

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This review provides systematic coverage of examples in the field of in-capillary electrophorecially mediated microanalysis (EMMA). The recent developments and applications in the time period up to mid 2011 have been described, as well as relevant older papers. The basic principles and modes of in-capillary assays have been demonstrated. An overview is also given of the various injection, separation and detection modes implemented in combination with EMMA. The review is presented in two parts mainly dealing with (i) enzymatic and (ii) derivatization or chemical reactions. Finally, the future trends of CE in performing and monitoring reactions have been drawn.

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Section: In‐capillary Assaymentioning

confidence: 99%

Recent developments and applications of EMMA in enzymatic and derivatization reactions

2011

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“…Observou-se que essa interação apresentou efeito negativo sobre o GH, deste modo, quando o pH aumentou para 8,5 e a relação E:S diminuiu para 1:150 o GH foi favorecido. Schuchert-Shi & Hauser (2009), ao trabalhar com tripsina, observaram que uma alta Tabela 2. Grau de hidrólise da caseína do leite de cabra da raça Moxotó em função das variáveis pH, temperatura, tempo de reação e relação enzima:substrato (E:S), com utilização da tripsina como enzima proteolítica.…”

Section: Hidrolisadounclassified

Avaliação de variáveis que influenciam a hidrólise enzimática da caseína do leite de cabra Moxotó

Lira

Bezerra

Silva

et al. 2010

Pesq. agropec. bras.

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Resumo − O objetivo deste trabalho foi avaliar os efeitos da temperatura, pH, relação enzima:substrato (E:S) e tempo de reação sobre a hidrólise enzimática da caseína do leite de cabra Moxotó, com uso de diferentes enzimas proteolíticas. A hidrólise enzimática da caseína caprina foi realizada com uso de tripsina, pepsina e papaína. Para cada enzima, foram utilizados dois níveis de cada variável, na avaliação de seus efeitos sobre a hidrólise da caseína, em um fatorial completo 2 4 . Os produtos da hidrólise foram visualizados em eletroforese SDS-PAGE. O melhor valor do grau de hidrólise com a enzima papaína foi obtido em pH 6,5, E:S de 1:150 e 5 horas de hidrólise a 50ºC (28,17%); com a tripsina, em pH 8,5, E:S de 1:150 e 5 horas a 40ºC (29,55%); e com a pepsina, em pH 3,0, E:S de 1:100 e 5 horas a 40ºC (38,27%). A hidrólise da caseína caprina é influenciada positivamente pelo pH e tempo de hidrólise, com o uso da pepsina. Foram observadas interações significativas antagônicas entre pH e relação E:S, com o uso da tripsina; e entre pH, temperatura e tempo de hidrólise, com o uso da papaína. A pepsina apresenta elevada eficiência na hidrólise de α s1 , β e κ-caseína, evidenciada por hidrolisados com massa molar abaixo de 14,4 kDa.Termos para indexação: Capra hircus, leite de cabra, papaína, pepsina, proteólise, tripsina. Evaluation of the variables that influence the enzymatic hydrolysis of Moxotó goat's milk caseinAbstract -The aim of this work was to evaluate the effects of temperature, pH, enzyme:substrate ratio (E:S), and reaction time on the enzymatic hydrolysis of Moxotó breed goat's milk casein, using different proteolytic enzymes. Enzymatic hydrolysis of the capric casein was performed using trypsin, pepsin and papain. For each enzyme, two levels of the variables were used to assess their effects on the hydrolysis of casein, using a full factorial design (2 4 ). The hydrolysis products were visualized on SDS-PAGE. The best hydrolysis degree using papain were at pH 6.5, E:S of 1:150 and 5 hours of hydrolysis at 50°C (28.17%); for trypsin, at pH 8.5, E:S of 1:150 and 5 hours at 40°C (29.55%); and for pepsin, at pH 3.0, E:S of 1:100 and 5 hours at 40°C (38.27%). Capric casein hydrolysis is affected positively by pH and reaction time, using pepsin. Significant antagonistic interactions were observed between pH and E:S, with trypsine; and between pH, temperature and reaction time, with papain. Pepsin has high α s1 , β e κ-casein hydrolysis efficiency, evidenced by the molar masses below 14.4 kDa of its products.

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“…For that reason, the reader is referred to the general recent reviews dealing with developments and applications of these detectors, such as electrochemical 185, 186, conductivity 187, 188, chemiluminescence 189, photoinduced chemiluminescence 190, electrochemiluminescence 191, mid‐infrared 192 and NMR 193, where few particular applications of these detectors for peptide CE analyses are mentioned. For example, C 4 D has been employed for the detection of CE separated peptic and tryptic peptide fragments of proteins (HSA, BSA and myoglobin) 194 and chemiluminescence detection based on reaction between hydrogen peroxide and N ‐(4‐aminobutyl)‐ N ‐ethylisoluminol‐tagged peptides in the presence of adenine as luminescence enhancer and Co 2+ ions as the catalyst was used for fast (2 min) microchip CE quantitation of carnosine‐related peptides in human CSF and canine plasma with LODs in the 28–34 nM range 195.…”

Section: Detectionmentioning

confidence: 99%

Recent developments in CE and CEC of peptides (2009–2011)

Kašička

2011

Electrophoresis

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The review brings a comprehensive survey of the recent developments of high-performance electroseparation methods in capillary and microchip formats: zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography and electrochromatography. Applications of these techniques to analysis, isolation, purification and physicochemical and biochemical characterization of peptides are described. Advances in the investigation of electromigration properties of peptides, and in the methodology of their analysis, such as sample preparation, adsorption suppression, EOF control and detection, are presented. New developments, in particular, CE and CEC modes are reported and several types of their applications to peptide analysis are described: conventional qualitative and quantitative analysis, determination in complex (bio)matrices, monitoring of chemical and enzymatical reactions and physical changes, amino acid, sequence and chiral analysis, and peptide mapping of proteins. Some micropreparative peptide separations are shown and capabilities of CE and CEC techniques to provide relevant physicochemical characteristics of peptides are demonstrated.

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Peptic and tryptic digestion of peptides and proteins monitored by capillary electrophoresis with contactless conductivity detection

Cited by 36 publications

References 41 publications

Recent developments and applications of EMMA in enzymatic and derivatization reactions

Recent developments and applications of EMMA in enzymatic and derivatization reactions

Avaliação de variáveis que influenciam a hidrólise enzimática da caseína do leite de cabra Moxotó

Recent developments in CE and CEC of peptides (2009–2011)

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