• Bulletin of the Korean Chemical Society
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• 제33권10호
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• pp.3233-3240
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• 2012

We demonstrated the combined applications of online protein digestion using trypsin immobilized enzyme reactor (IMER) and dual tandem mass spectrometry with collisionally activated dissociation (CAD) and electron transfer dissociation (ETD) for tryptic peptides eluted through the trypsin-IMER. For the trypsin-IMER, the organic and inorganic hybrid monolithic material was used. By employing the trypsin-IMER, the long digestion time could be saved with little or no sacrifice of the digestion efficiency, which was demonstrated for standard protein samples. For three model proteins (cytochrome c, carbonic anhydrase, and bovine serum albumin), the tryptic peptides digested by the IMER were analyzed using LC-MS/MS with the dual application of CAD and ETD. As previously shown by others, the dual application of CAD and ETD increased the sequence coverage in comparison with CAD application only. In particular, ETD was very useful for the analysis of highly-protontated peptide cations, e.g., ${\geq}3+$. The combination approach provided the advantages of both trypsin-IMER and CAD/ETD dual tandem mass spectrometry applications, which are rapid digestion (i.e., 10 min), good digestion efficiency, online coupling of trypsin-IMER and liquid chromatography, and high sequence coverage.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• 제16권3호
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• pp.417-424
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• 2003

Inhibitory activities against angiotensin I-converting enzyme (ACE) of enzymatic hydrolysates of porcine skeletal muscle proteins were investigated. Myosin B, myosin, actin, tropomyosin, troponin and water-soluble proteins extracted from pork loin were digested by eight kinds of proteases, including pepsin, $\alpha$-chymotrypsin, and trypsin. After digestion, hydrolysates produced from all proteins showed ACE inhibitory activities, and the peptic hydrolysate showed the strongest activity. In the case of myosin B, the molar concentration of peptic hydrolysate required to inhibit 50% of the activity increased gradually as digestion proceeded. The hydrolysates produced by sequential digestion with pepsin and $\alpha$-chymotrypsin, pepsin and trypsin or pepsin and pancreatin showed weaker activities than those by pepsin alone, suggesting that ACE inhibitory peptides from peptic digestion might lose their active sequences after digestion by the second protease. However, the hydrolysates produced by sequential digestion showed stronger activities than those by $\alpha$-chymotrypsin, trypsin or pancreatin alone. These results suggested that the hydrolysates of porcine meat were able to show ACE inhibitory activity, even if they were digested in vivo, and that pork might be a useful source of physiologically functional factors.

• Asian-Australasian Journal of Animal Sciences
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• 제6권2호
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• pp.319-323
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• 1993

The effect of bovine growth hormone digested with trypsin on whole-body protein synthesis in vitro of chicken embryos was investigated by using a whole-embryo culture system. Bovine growth hormone at 5.3 and 530 ng/ml was digested partially and completely with trypsin for 4 min and 18 h, respectively. After culturing chicken embryos with a synthetic medium containing $L-[4-^3H]$ pheylalanine, whole-embryo protein synthesis was determined from the ratio of specific radioactivities of free and protein-bound pheylalanine. Whole-embryo protein synthesis of the control group cultured with no bovine growth hormone was $49.5{\pm}2.2%/d$. There was no significant interaction between digestion time and the concentration of trypsin-digested bovine growth hormone. Tryptic digestion of bovine growth hormone increased fractional synthesis rates of whole-body protein compared to the 0-min groups, and there was no significant difference between the 4-min and 18-h groups. The higher concentration (530 ng/ml) of trypsin-digested bovine growth hormone was more effective in enhancing whole-embryo protein synthesis than the lower concentration (5.3 ng/ml).

• Mass Spectrometry Letters
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• 제15권1호
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• pp.62-68
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• 2024

The Suspension Trapping (S-Trap) method has been a prominent sample preparation technique since its introduction in 2014. Its capacity to induce protein aggregation using organic solvents has significantly improved protein purification and facilitated peptide identification. However, its full potential for automation has been limited by the lack of a suitable liquid handling system until recently. In this study, we aimed to enhance the automation of S-Trap sample preparation by optimizing the S-Trap digestion process, incorporating triethylammonium bicarbonate (TEAB) and CaCl₂. The utilization of TEAB buffer conditions in this innovative process led to a noteworthy 12% improvement in protein identification. Additionally, through careful observation of various incubation conditions, we streamlined the entire sample preparation workflow into a concise 4 hours timeline, covering reduction, alkylation, and trypsin incubation stages. This refined and expedited automated S-Trap digestion process not only showcased exceptional time efficiency but also improved trypsin digestion, resulting in increased protein identification.

• Molecules and Cells
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• 제23권3호
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• pp.340-348
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• 2007

Although considerable effort has been devoted in the mass spectrometric analysis of phosphorylated peptides, successful identification of multi-phosphorylated peptides in enzymatically digested protein samples still remains challenging. The ionization behavior of multi-phosphorylated peptides appears to be somewhat different from that of mono- or di-phosphorylated peptides. In this study, we demonstrate increased sensitivity of detection of multi-phosphorylated peptides of beta casein without using phosphopeptide enrichment techniques. Proteinase K digestion alone increased the detection limit of beta casein multi-phosphorylated peptides in the LC-MS analysis almost 500 fold, compared to conventional trypsin digestion (~50 pmol). In order to understand this effect, various factors affecting the ionization of phosphopeptides were investigated. Unlike ionizations of phosphopeptides with minor modifications, those of multi-phosphorylated peptides appeared to be subject to effects such as selectively suppressed ionization by more ionizable peptides and decreased ionization efficiency by multi-phosphorylation. The enhanced detection limit of multi-phosphorylated peptides resulting from proteinase K digestion was validated using a complex protein sample, namely a lysate of HEK 293 cells. Compared to trypsin digestion, the numbers of phosphopeptides identified and modification sites per peptide were noticeably increased by proteinase K digestion. Non-specific proteases such as proteinase K and elastase have been used in the past to increase detection of phosphorylation sites but the effectiveness of proteinase K digestion for multi-phosphorylated peptides has not been reported.

• 대한수의학회지
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• 제6권1호
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• pp.10-17
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• 1966

Salmonella pullorum 표준형균주(標準型菌株) 및 변이형균주(變異型菌株)의 균체물질(菌體物質)을 가열추출(加熱抽出), 산성추출(酸性抽出), 산성가열추출(酸性加熱抽出), trypsin 소화(消化) 급류산(及硫酸) ammonium 포화(飽和)등의 방법(方法)으로 처리(處理)하여 얻은 각(各) 분층(分層)의 항원성(抗原性)을 혈구응집반응(血球凝集反應) 및 침강반응(沈降反應)으로 비교검토(比較檢討)하여 다음과 같은 결과(結果)를 얻었다. 1. S. pullorum 균체항원(菌體抗原)은 표준형균주(標準型菌株) 및 변이형균주(變異型菌株) 다같이 가열추출(加熱抽出)에서만이 인(人)0형혈구(型血球)와 계혈구(鷄血球)에 흡착(吸着)되어 혈구응집반응(血球凝集反應)의 반응(反應)을 나타내였다. 2. Trypsin의 영향(影響)은 별(別)로 저명(著明)하지는 아니하나 산성추출항원(酸性抽出抗原)에서 표준형균주(標準型菌株)보다 변이형균주(變異型菌株)가 혈구응집반응(血球凝集反應)에서 약간(若干) 양성(陽性)으로 변전(變轉)되었다. 3. S. pullorum 표준형균주(標準型菌株)의 항원성물질(抗原性物質)은 유안포화시(硫安飽和時) 거의 그 침전분층(沈澱分層)에 이행(移行)되며 이는 동(同) 침전분층(沈澱分層)을 trypsin 소화(消化)하므로써 혈구응집반응(血球凝集反應)에 강력(强力)한 항원성(抗原性)을 보유(保有)하고 있으므로 증명(證明)할 수 있었다. 4. 유안포화시(硫安飽和時) S. pullorum 변이형균주(變移型菌株)는 산성추출항원(酸性抽出抗原)에서는 침전분층(沈澱分層)을 얻을 수 없었고 그 상청(上淸)에는 항원성(抗原性)도 없었으나 가열추출항원(加熱抽出抗原)에 있어서는 상청(上淸) 급(及) 침전분층(沈澱分層) 공(共)히 혈구응집반응(血球凝集反應)에 양성(陽性)을 나타내었다. 5. 각종추출항원분층(各種抽出抗原分層)의 침강반응(沈降反應)에서는 거의 반응(反應)을 나타내지 아니하였다.

• Bulletin of the Korean Chemical Society
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• 제32권2호
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• pp.599-604
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• 2011

In typical proteomic analysis, trypsin digestion is one of the most time-consuming steps. Conventional proteomic sample preparation methods use an overnight trypsin digestion method. In this study, we compared high-pressure cycling technology (PCT) during enzyme digestion for proteome analysis to the conventional method. We examined the effect of PCT on enzyme activity at temperatures of 25, 37, and $50^{\circ}C$. Although a fast digestion (1 h) was used for the standard protein mixture analysis, the PCT-assisted method with urea showed better results for protein sequence coverage and the number of peptides identified compared with the conventional method. There was no significant difference between temperatures for PCT-assisted digestion; however, we selected PCT-assisted digestion with urea at $25^{\circ}C$ as an optimized method for fast enzyme digestion, based on peptide carbamylation at these conditions. The optimized method was used for stem cell proteome analysis. We identified 233, 264 and 137 proteins using the conventional method with urea at $37^{\circ}C$ for 16h, the PCT-assisted digestion with urea at $25^{\circ}C$ for 1 h, and the non-PCT-assisted digestion with urea at $25^{\circ}C$ for 1 h, respectively. A comparison of these results suggests that PCT enhanced the enzyme digestion by permitting better access to cleavage sites on the proteins.

• 한국양식학회지
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• 제5권1호
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• pp.1-7
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• 1992

어분의 대체원료로써 대두당의 유효성을 개선하기 위하여, 사료용 대두정 (H-SBM)과 식품용 미처리 대두당(R-SBM)을 Ex 처리하여, 무지개송어에 있어서 사료중 대두박의 단백질 및 탄수화물의 소화율을 측정하였으며, 트립신 저해 인자 (TI)의 활성과 단백질 소화율과의 관계에 대해서 검토하였다. 그 결과 H-SBM 및 R-SBM 모두 Ex 처리함으로써 단백질의 소화율이 $95{\%}$까지 개선되었다. 한편, R-SBM의 탄수화물의 소화율은 Ex 처리에 의해 향상되었으나, H-SBM는 개선되지 않았다. 또 Ex 처리에 의해 대두박중의 TI 활성은 대두분 소실되었으며, R-SBM에는 TI의 소실화에 의해 단백질의 소화율이 향상되었으나, 이것이 Ex 처리에 의한 TI의 활성의 소실에 의한 것인지는 본 실험 조건 하에서는 명백히 할 수 없었으며, 금후 구체적인 검토를 필요로 한다.

• 한국식품과학회지
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• 제18권5호
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• pp.339-344
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• 1986

Trypsin inhibitor가 trypsin과 안정한 복합체를 형성하고 동시에 일반 단백질은 trypsin에 의하여 가수분해 되는 원리를 이용하여 trypsin inhibitor를 용이하게 검정 할 수 있는 방법을 고안하였다. trypsin으로 가수분해 시킨 대두추출액을 Sephadex G-50을 이용하여 trypsin-trypsin inhibitor 복합체를 분리시킨 후에 SDS 전기 영동으로 trypsin inhibitor를 복합체를 분리시킨 후에 SDS 전기 영동으로 trypsin inhibitor를 검정할 수 있었다. 이들 trypsin inhibitor는 trypsin에 의한 2차 가수분해에서도 가수분해 되지 않았으며, 또한 2차원 전기영동과 DEAE-Sephades A-25크로마토 그래피를 이용하여 trypsin inhibitor가 trypsin과 복합체를 형성하는 능력을 검정함으로써 본 방법의 유효성을 확인하였다. 본 실험 방법으로 대두(Hill 품종)의 trypsin inhibitor를 검정한 결과 7개의 trypsin inhibitor를 찾아 낼 수 있었다.