目的:探索胶上蛋白原位酶切、肽质量指纹图(PMF)分析以及数据库检索方法的优化条件,建立适合于亚细胞比较蛋白质组学研究的质谱鉴定策略。方法:从考马斯亮蓝染色的2-DE胶上取下牛血清白蛋白(BSA)标准蛋白进行胶内原位酶切,与基质(CHCA)...目的:探索胶上蛋白原位酶切、肽质量指纹图(PMF)分析以及数据库检索方法的优化条件,建立适合于亚细胞比较蛋白质组学研究的质谱鉴定策略。方法:从考马斯亮蓝染色的2-DE胶上取下牛血清白蛋白(BSA)标准蛋白进行胶内原位酶切,与基质(CHCA)混匀后进行MALDI-TOF MS分析;对来源于人肝细胞癌细胞株QGY-7703线粒体的差异表达的候选蛋白点L9,按照通过BSA标准蛋白优化的条件进行MALDI-TOF质谱鉴定;其肽质量指纹图(PMF)经MS-Fit检索。结果:经数据库检索,BSA的肽质量指纹图(PMF)匹配肽质量数为10/11,序列覆盖率为19%,说明实验条件完全可信。候选蛋白点L9的PMF数据经数据库检索后,排名前两位的蛋白均为OXCT(3-oxoacid CoA transferase 1 precursor),其匹配肽段为9/13,序列覆盖率为24%;且OXCT的理论分子量(56kda),理论等电点(7.1),与胶上L9蛋白的位置相符。故候选蛋白点L9确认为OXCT(3-oxoacid CoA transferase 1 precursor)。结论:本文所确定的鉴定策略适用于线粒体比较蛋白质组学的研究。展开更多
目的:为了优化肽质量指纹谱(Peptide mass fingerprinting,PMF)鉴定蛋白质时采用MS-Fit引擎搜索的分析参数。方法:将2-DE分离后的Carbonic anhydrase-2和BSA进行胶内充分酶解,肽段经过MALDI-TOF-MS分析得到PMF数据。选择Swissprot数据库...目的:为了优化肽质量指纹谱(Peptide mass fingerprinting,PMF)鉴定蛋白质时采用MS-Fit引擎搜索的分析参数。方法:将2-DE分离后的Carbonic anhydrase-2和BSA进行胶内充分酶解,肽段经过MALDI-TOF-MS分析得到PMF数据。选择Swissprot数据库,以Carbonic anhydrase2为模型优化搜索参数。结果:主要搜索参数的最佳设置为:半胱氨酸修饰为Carbamidomethylation,肽质量容错模型为百分数,在研究中0.1%容错数为最好。结论:本文通过标准蛋白对搜索主要参数的优化,建立了方便、可靠的MS-Fit搜索参数模式。展开更多
Transferrin（TF） and its isoforms have been widely reported via various analytical techniques, including a noticeable increased number of isoforms with low content of sialic acid（asialo-, monosialo-, and disialo-tra...Transferrin（TF） and its isoforms have been widely reported via various analytical techniques, including a noticeable increased number of isoforms with low content of sialic acid（asialo-, monosialo-, and disialo-transferrin） and asialo-TF as well as disialo-TF, with one or several oligosaccharides released in human serum transferrin（hTf）. Here, hTf has been purified by native gradient polyacrylamide gel electrophoresis（PAGEso） before use. The hTf extracted with the electron-transfer approach showed a single subunit band（77.1 Da） in the SDS-PAGE gel, but it exhibited two bands in the native and denatured isoelectric focusing（IEF） gels, namely, hTf-2Fe^3＋ and apo-hTf, without finding any other transferrin isoforms. A reversed phase HPLC（RP-HPLC） equipped with a C18 column effectively separated hTf and its polymers and combined off-line techniques, including peptide mass fingerprinting（PMF）, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（MALDI-TOF-MS） and database search, and identified the high homology among hTf, apo-hTf, and their isoforms. Moreover, the elution solution consisting of acetonitrile and formic acid could easily denature both hTf and apo-hTf to form various isoforms during separation with HPLC, indicating that chemical factors lead to the formation of various isoforms in transferrin, artificially, during extraction and separation. The authors claimed that only two transferrin isoforms existed in the NHS, namely, hTf-2Fe^3＋ and apo-hTf, which could be employed in biomarkers, to distinguish the healthy population from many disease sufferers, such as, carbohydrate-deficient transferrin（CDT）展开更多
文摘目的:探索胶上蛋白原位酶切、肽质量指纹图(PMF)分析以及数据库检索方法的优化条件,建立适合于亚细胞比较蛋白质组学研究的质谱鉴定策略。方法:从考马斯亮蓝染色的2-DE胶上取下牛血清白蛋白(BSA)标准蛋白进行胶内原位酶切,与基质(CHCA)混匀后进行MALDI-TOF MS分析;对来源于人肝细胞癌细胞株QGY-7703线粒体的差异表达的候选蛋白点L9,按照通过BSA标准蛋白优化的条件进行MALDI-TOF质谱鉴定;其肽质量指纹图(PMF)经MS-Fit检索。结果:经数据库检索,BSA的肽质量指纹图(PMF)匹配肽质量数为10/11,序列覆盖率为19%,说明实验条件完全可信。候选蛋白点L9的PMF数据经数据库检索后,排名前两位的蛋白均为OXCT(3-oxoacid CoA transferase 1 precursor),其匹配肽段为9/13,序列覆盖率为24%;且OXCT的理论分子量(56kda),理论等电点(7.1),与胶上L9蛋白的位置相符。故候选蛋白点L9确认为OXCT(3-oxoacid CoA transferase 1 precursor)。结论:本文所确定的鉴定策略适用于线粒体比较蛋白质组学的研究。
基金This work was supported by Teaching and Research Award Program for Outstanding Young Teachers in Higher Education Institu-tions by Ministry of Education of China, the National Natural Science Foundation (No. 30370783), and the Key Project of Science and Technology by Heilongjiang Province (No. GB03C601-1).
文摘目的:为了优化肽质量指纹谱(Peptide mass fingerprinting,PMF)鉴定蛋白质时采用MS-Fit引擎搜索的分析参数。方法:将2-DE分离后的Carbonic anhydrase-2和BSA进行胶内充分酶解,肽段经过MALDI-TOF-MS分析得到PMF数据。选择Swissprot数据库,以Carbonic anhydrase2为模型优化搜索参数。结果:主要搜索参数的最佳设置为:半胱氨酸修饰为Carbamidomethylation,肽质量容错模型为百分数,在研究中0.1%容错数为最好。结论:本文通过标准蛋白对搜索主要参数的优化,建立了方便、可靠的MS-Fit搜索参数模式。
基金National Natural Science Foundation of China(No.30470372Foundation of Xiamen Natural Science of China(No.3502Z2001262)Program for Innovative Research Team in Science and Technology in Fujian Province University
文摘Transferrin（TF） and its isoforms have been widely reported via various analytical techniques, including a noticeable increased number of isoforms with low content of sialic acid（asialo-, monosialo-, and disialo-transferrin） and asialo-TF as well as disialo-TF, with one or several oligosaccharides released in human serum transferrin（hTf）. Here, hTf has been purified by native gradient polyacrylamide gel electrophoresis（PAGEso） before use. The hTf extracted with the electron-transfer approach showed a single subunit band（77.1 Da） in the SDS-PAGE gel, but it exhibited two bands in the native and denatured isoelectric focusing（IEF） gels, namely, hTf-2Fe^3＋ and apo-hTf, without finding any other transferrin isoforms. A reversed phase HPLC（RP-HPLC） equipped with a C18 column effectively separated hTf and its polymers and combined off-line techniques, including peptide mass fingerprinting（PMF）, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（MALDI-TOF-MS） and database search, and identified the high homology among hTf, apo-hTf, and their isoforms. Moreover, the elution solution consisting of acetonitrile and formic acid could easily denature both hTf and apo-hTf to form various isoforms during separation with HPLC, indicating that chemical factors lead to the formation of various isoforms in transferrin, artificially, during extraction and separation. The authors claimed that only two transferrin isoforms existed in the NHS, namely, hTf-2Fe^3＋ and apo-hTf, which could be employed in biomarkers, to distinguish the healthy population from many disease sufferers, such as, carbohydrate-deficient transferrin（CDT）
