使用肽指纹图谱进行数据库搜索----示意
丁香园论坛
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:)刚才在精华版看到一些朋友问到在用MALDI测得肽段分子量后怎么进行数据库搜索的问题，觉得有必要给新手们详细阐明一下，所以贴出了我自己硕士毕业论文中的相关部分与大家共享，也请高手批评指正。我的样品种属是小鼠，我举例的这个SPOT没有鉴定出来，呵呵。
2．蛋白斑点PRO2
Fig.9 peptide mass fingerprint(PMF) of PRO2 digested by trypsin
利用MASCOT程序进行检索
输入肽段质量数为：935.49 1570.91 1578.89 1641.88 1758.98 1994.08 2558.37 2581.36 2594.50 2597.50 2722.33
检索参数
Database searched SWISSPROTTaxonomy Mus musculusEnzyme trypsinMax missed cleavages 1 or 2Fixed modifications are CarbamidomethylProtein mass NonePeptide mass tolerance ±0.5 DaMass values are monoisotopicReport top 20
搜索结果如下：
（1） gi|20894496 Mass: 41060 Total score: 44 Peptides matched: 4 hypothetical protein XP_156355 [Mus musculus]
gi|284950 Mass: 10568 Total score: 27 Peptides matched: 2 Ig kappa chain V region - mouse
（2 ）gi|20820877 Mass: 16730 Total score: 44 Peptides matched: 3 hypothetical protein XP_149891 [Mus musculus]
（3 ）gi|28523811 Mass: 34454 Total score: 38 Peptides matched: 3 similar to protease [Mus musculus]
gi|20833889 Mass: 12794 Total score: 32 Peptides matched: 2 hypothetical protein XP_150900 [Mus musculus]
gi|20911605 Mass: 11530 Total score: 32 Peptides matched: 2 hypothetical protein XP_158628 [Mus musculus]
gi|20820603 Mass: 15669 Total score: 31 Peptides matched: 2 hypothetical protein XP_158099 [Mus musculus]
gi|28482850 Mass: 18455 Total score: 28 Peptides matched: 2 hypothetical protein XP_283657 [Mus musculus]
gi|26353630 Mass: 19939 Total score: 28 Peptides matched: 2
换PeptIdent程序进行检索，参数如下
Parameters Parameters set
Database searched SWISS-PROTProtein mass range 40-60KDaPI range 4-6Species searched Mus musculusDigested used TrypsinPeptide mass accurance ±0.5DaCysteines are treated with IdoacetamidePeptide masses are monoisotopicNumber of peptides required for match 4Number of missed cleavage sites 1Number of matching proteins display 20
结果如下：
Score peptide matches AC ID Description pI Mw
（1）0.64 7 Q02566 MYH6_MOUSE Myosin heavy chain, cardiac muscle alpha isoform (MyHC-alpha). - Mus musculus (Mouse). 5.57 223565.34
（2）0.64 7 P97313 PRKD_MOUSE DNA-dependent protein kinase catalytic subunit (EC 2.7.1.37) (DNA- PKcs) (P460). - Mus musculus (Mouse). 6.70 471387.70
（3）0.55 6 Q61555 FBN2_MOUSE_1 CHAIN 1: FIBRILLIN 2. - Mus musculus (Mouse). 4.71 310393.74
（4）0.45 5 P97929 BRC2_MOUSE Breast cancer type 2 susceptibility protein. - Mus musculus (Mouse). 6.23 370663.75
（5）0.45 5 P59242 CING_MOUSE Cingulin. - Mus musculus (Mouse). 5.72 136446.87
在NCBInr数据库中，检索到的蛋白没有一个MOWSE分值在62分以上（p﹤0.05），最高分值仅为44分，且为一假设蛋白，故不能得出正确结果。换SWISSPROT数据库检索，分值最高的Adseverin (Scinderin) (Gelsolin-like protein)为37分，也不能正确鉴定。PeptIdent所检索到的Q02566 MYH6_MOUSE Myosin heavy chain, cardiac muscle alpha isoform (MyHC-alpha). - Mus musculus (Mouse).虽然匹配肽段数达到了7个，但由于序列覆盖率仅为6.8%，仍然得不到正确鉴定。综合上述结果，不能鉴定PRO2的蛋白质种属。
2．蛋白斑点PRO2
Fig.9 peptide mass fingerprint(PMF) of PRO2 digested by trypsin
利用MASCOT程序进行检索
输入肽段质量数为：935.49 1570.91 1578.89 1641.88 1758.98 1994.08 2558.37 2581.36 2594.50 2597.50 2722.33
检索参数
Database searched SWISSPROTTaxonomy Mus musculusEnzyme trypsinMax missed cleavages 1 or 2Fixed modifications are CarbamidomethylProtein mass NonePeptide mass tolerance ±0.5 DaMass values are monoisotopicReport top 20
搜索结果如下：
（1） gi|20894496 Mass: 41060 Total score: 44 Peptides matched: 4 hypothetical protein XP_156355 [Mus musculus]
gi|284950 Mass: 10568 Total score: 27 Peptides matched: 2 Ig kappa chain V region - mouse
（2 ）gi|20820877 Mass: 16730 Total score: 44 Peptides matched: 3 hypothetical protein XP_149891 [Mus musculus]
（3 ）gi|28523811 Mass: 34454 Total score: 38 Peptides matched: 3 similar to protease [Mus musculus]
gi|20833889 Mass: 12794 Total score: 32 Peptides matched: 2 hypothetical protein XP_150900 [Mus musculus]
gi|20911605 Mass: 11530 Total score: 32 Peptides matched: 2 hypothetical protein XP_158628 [Mus musculus]
gi|20820603 Mass: 15669 Total score: 31 Peptides matched: 2 hypothetical protein XP_158099 [Mus musculus]
gi|28482850 Mass: 18455 Total score: 28 Peptides matched: 2 hypothetical protein XP_283657 [Mus musculus]
gi|26353630 Mass: 19939 Total score: 28 Peptides matched: 2
换PeptIdent程序进行检索，参数如下
Parameters Parameters set
Database searched SWISS-PROTProtein mass range 40-60KDaPI range 4-6Species searched Mus musculusDigested used TrypsinPeptide mass accurance ±0.5DaCysteines are treated with IdoacetamidePeptide masses are monoisotopicNumber of peptides required for match 4Number of missed cleavage sites 1Number of matching proteins display 20
结果如下：
Score peptide matches AC ID Description pI Mw
（1）0.64 7 Q02566 MYH6_MOUSE Myosin heavy chain, cardiac muscle alpha isoform (MyHC-alpha). - Mus musculus (Mouse). 5.57 223565.34
（2）0.64 7 P97313 PRKD_MOUSE DNA-dependent protein kinase catalytic subunit (EC 2.7.1.37) (DNA- PKcs) (P460). - Mus musculus (Mouse). 6.70 471387.70
（3）0.55 6 Q61555 FBN2_MOUSE_1 CHAIN 1: FIBRILLIN 2. - Mus musculus (Mouse). 4.71 310393.74
（4）0.45 5 P97929 BRC2_MOUSE Breast cancer type 2 susceptibility protein. - Mus musculus (Mouse). 6.23 370663.75
（5）0.45 5 P59242 CING_MOUSE Cingulin. - Mus musculus (Mouse). 5.72 136446.87
在NCBInr数据库中，检索到的蛋白没有一个MOWSE分值在62分以上（p﹤0.05），最高分值仅为44分，且为一假设蛋白，故不能得出正确结果。换SWISSPROT数据库检索，分值最高的Adseverin (Scinderin) (Gelsolin-like protein)为37分，也不能正确鉴定。PeptIdent所检索到的Q02566 MYH6_MOUSE Myosin heavy chain, cardiac muscle alpha isoform (MyHC-alpha). - Mus musculus (Mouse).虽然匹配肽段数达到了7个，但由于序列覆盖率仅为6.8%，仍然得不到正确鉴定。综合上述结果，不能鉴定PRO2的蛋白质种属。
