• Bulletin of the Korean Chemical Society
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• 제31권6호
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• pp.1527-1534
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• 2010

The conventional peptide modification process of guanidination, in which the amino groups of lysine residues are converted to guanidino groups using O-methylisourea to create more basic homoarginine residues, is often used to improve the signal intensity of lysine-containing peptides in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Here, we used three different protease enzymes (trypsin, Lys-C, and Glu-C) to evaluate the effects of guanidination on the MS signals of two enzymatically digested proteins. Horse heart myoglobin and bovine serum albumin were guanidinated either before or after digestion with trypsin, Lys-C, or Glu-C. The resulting peptides were subjected to MALDI-MS, and signal intensities and sequence coverage were systematically evaluated for each digest. Guanidination prior to Glu-C digestion improved sequence coverage for both proteins. For myoglobin, guanidination before enzymatic digestion with trypsin or Lys-C also enhanced sequence coverage, but guanidination after enzymatic digestion enhanced sequence coverage only with Lys-C. For albumin, guanidination either before or after Glu-C digestion increased sequence coverage, whereas pre- or post-digestion guanidination decreased sequence coverage with trypsin and Lys-C. The amino acid composition of a protein appears to be the major factor determining whether guanidination will enhance its MALDI-MS sequence coverage.

• 분석과학
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• 제25권2호
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• pp.114-120
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• 2012

펩타이드의 정성 분석에서, O-Methylisourea는 펩타이드의 특정 아미노산(예. 라이신)에 화합결합하여 해당 펩타이드의 신호를 증진시키기 때문에 펩타이드를 matrix-assisted laser desorption/ionizationmass spectrometry (MALDI-MS) 분석하기 위해 흔히 사용되는데, 이러한 과정은 guanidination이라고 불린다. Guanidination은 반응 조건에 따라 효율이 변하게 된다. 본 연구에서는 트립신으로 가수분해된 미오글로빈 단백질을 세 가지 다른 반응시약 (O-methylisourea, S-methylisothiourea, 2-methyl-2-imidazoline)을 사용하여 $65^{\circ}C$ 에서 1 시간 동안 다양한 pH 조건 (pH 4.0, 7.0 및 10.5)에서 guanidination 반응을 수행하였는데, 실험 결과 O-methylisourea와 pH 10.5이 가장 좋은 효율을 나타내었다. 다음으로 O-methylisourea와 pH 10.5의 반응 조건을 이용하여 열, 마이크로파, 초음파 등과 같은 다양한 조건에서 시간을 변화시켜 가면서 guanidination을 연구하였는데, 열을 이용하여 60 분 동안 반응시키는 것이 가장 효과적이었다. 결론적으로 O-methylisourea을 이용하여 pH 10.5 용액에서 열을 이용하여 1 시간 동안 $65^{\circ}C$에서 가열하는 것이 guanidination을 위한 최적의 조건이었다.

• Preventive Nutrition and Food Science
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• 제1권1호
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• pp.10-15
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• 1996

To determine the quality of heated protein, in vitro method, invluding lysine, lysionalanine, and fructose-lysine as well as homoarginine by guanidination of lysine, was assessed using heated casein with of without glucose. In vivo methods such as PER, digestibility and BV were also tried on homoarginine, lysinoalanine, fructoselysine, and lysine. The nonreactive lysine for huanidination was hardly digestive, while the non heat damaged lysine side chanis in the protein were accessible for guanidination as well as for the digestion. A linear correlation(${\gamma}$=0.80) was obstained between PER and digestibility of the analysed lysine. Digestibility of homoarginine was higher that of true protein. However, in the guanidinated heated casein with glucose, digestibility of homoarginine was significantly reduced. It is suggested that the homoarginine method may mislead to over- or underestimation of the damaged protein quality.