지원사업
학술연구/단체지원/교육 등 연구자 활동을 지속하도록 DBpia가 지원하고 있어요.
커뮤니티
연구자들이 자신의 연구와 전문성을 널리 알리고, 새로운 협력의 기회를 만들 수 있는 네트워킹 공간이에요.
논문 기본 정보
- 자료유형
- 학술저널
- 저자정보
- 발행연도
- 2022.12
- 수록면
- 184 - 188 (5page)
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초록· 키워드
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Mass spectrometry (MS) is the cutting-edge platform that propels biological and clinical research. MS-based bottom-up proteomics has expedited the characterization of signal transductions and aberrant proteomes of disease specimens through protein identification, protein-protein interaction discovery, and post-translational dynamics profiling. The typical pipeline of the bottom-up strategy for MS involves protein digestion prerequisite to peptide separation by liquid chromatography and data acquisition on a mass spectrometer.<SUP>1</SUP> The refinement of the sample processing is critical to fulfilling the quest for augmenting proteome coverage, robustness, and throughput, alongside the quantum leap of the instrument and computational analysis.
Enzymatic digestion of proteomes into peptides is a hallmark of bottom-up proteomics, influencing the quality of protein identification and quantitation. To deduce the optimal condition for protease activity, many studies have been conducted using different lysis reagents and buffers.<SUP>2-5</SUP> The conventional in-solution digestion method using urea is the most feasible and cost-effective. Urea is a chaotropic denaturant that can disrupt intraprotein interactions, resulting in protein unfolding to facilitate trypsin accessibility. As a hydrophilic compound, urea can be removed by reversed-phase liquid chromatography (RPHPLC), hence its compatibility with mass spectrometry. However, carbamoylation modification on proteins and peptides’ amines caused by urea under heat hinders digestion sites.<SUP>6,7</SUP> Sodium dodecyl sulfate (SDS) is an alternative to urea with more efficient solubilization of membrane proteins, but it can damage the RP-HPLC. Evidence and concerns were also raised regarding the ion suppression and poor spectra in electrospray ionization mass spectrometry, which resulted from residual SDS exceeding 0.01%.<SUP>8,9</SUP> To resolve this, filter-aided sample preparation (FASP) was established to remove the ionic detergent by centrifugation and retain proteins above the molecular weight cut-off.<SUP>3,5</SUP> Another imperative method is the in-gel digestion approach that utilizes size-based separation assisted by negatively charged protein-SDS complexes. Not only does it dissipate SDS during electrophoresis, but it also enhances the depth of proteome characterization by reducing sample complexity.<SUP>10,11</SUP>
DataOn is a web-based resource established by Korea Institute of Science and Technology Information to improve re-analysis of registered data. In this protocol, we exemplified in-gel, in-solution and FASP digestion strategies. The demonstration videos entitled “Comparative sample preparation methods for label-free proteomic analysis (videos)” can be accessed at: https://doi.org/ 10.22711/idr/966, and through Supplementary Materials.
Enzymatic digestion of proteomes into peptides is a hallmark of bottom-up proteomics, influencing the quality of protein identification and quantitation. To deduce the optimal condition for protease activity, many studies have been conducted using different lysis reagents and buffers.<SUP>2-5</SUP> The conventional in-solution digestion method using urea is the most feasible and cost-effective. Urea is a chaotropic denaturant that can disrupt intraprotein interactions, resulting in protein unfolding to facilitate trypsin accessibility. As a hydrophilic compound, urea can be removed by reversed-phase liquid chromatography (RPHPLC), hence its compatibility with mass spectrometry. However, carbamoylation modification on proteins and peptides’ amines caused by urea under heat hinders digestion sites.<SUP>6,7</SUP> Sodium dodecyl sulfate (SDS) is an alternative to urea with more efficient solubilization of membrane proteins, but it can damage the RP-HPLC. Evidence and concerns were also raised regarding the ion suppression and poor spectra in electrospray ionization mass spectrometry, which resulted from residual SDS exceeding 0.01%.<SUP>8,9</SUP> To resolve this, filter-aided sample preparation (FASP) was established to remove the ionic detergent by centrifugation and retain proteins above the molecular weight cut-off.<SUP>3,5</SUP> Another imperative method is the in-gel digestion approach that utilizes size-based separation assisted by negatively charged protein-SDS complexes. Not only does it dissipate SDS during electrophoresis, but it also enhances the depth of proteome characterization by reducing sample complexity.<SUP>10,11</SUP>
DataOn is a web-based resource established by Korea Institute of Science and Technology Information to improve re-analysis of registered data. In this protocol, we exemplified in-gel, in-solution and FASP digestion strategies. The demonstration videos entitled “Comparative sample preparation methods for label-free proteomic analysis (videos)” can be accessed at: https://doi.org/ 10.22711/idr/966, and through Supplementary Materials.
목차
Procedure
In-solution digestion
FASP
Discussion
References
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