w********e
发帖数: 275 | 1 I'm not familiar with protein mass spec,I'd appreciate some help for a
question.
The protein samples was digested by trypsin, which cuts after K and R. But
why some peptide fragments follow amino acids other than K and R in the
result file? and the same fragment appears more then once. are those
fragments real?
Thanks a lot! |
o**4
发帖数: 35028 | 2 之后蛋白片段好像还是要被电子束什么的再打碎一次，所以测序不一定是peptide的第
一个氨基酸开始的
【在 w********e 的大作中提到】
: I'm not familiar with protein mass spec,I'd appreciate some help for a
: question.
: The protein samples was digested by trypsin, which cuts after K and R. But
: why some peptide fragments follow amino acids other than K and R in the
: result file? and the same fragment appears more then once. are those
: fragments real?
: Thanks a lot!
|
w********e
发帖数: 275 | 3 thanks for the information! so do you mean that those fragments are as real
as the fragments following K and R?
Thanks!
【在 o**4 的大作中提到】
: 之后蛋白片段好像还是要被电子束什么的再打碎一次，所以测序不一定是peptide的第
: 一个氨基酸开始的
|
o**4
发帖数: 35028 | 4 我觉得是，
real
【在 w********e 的大作中提到】
: thanks for the information! so do you mean that those fragments are as real
: as the fragments following K and R?
: Thanks!
|
l****n
发帖数: 711 | 5 那些peptide是non-specific cleavage引起的，这个很常见，能占到快20%，搜索的时
候一般都会包括这些partial cleavage的peptides。peptide的两端里有一端是non-
specific cleavage问题不大。可信不可信主要看score， 和重复出现的次数没什么关
系。
【在 w********e 的大作中提到】
: I'm not familiar with protein mass spec,I'd appreciate some help for a
: question.
: The protein samples was digested by trypsin, which cuts after K and R. But
: why some peptide fragments follow amino acids other than K and R in the
: result file? and the same fragment appears more then once. are those
: fragments real?
: Thanks a lot!
|
w********e
发帖数: 275 | 6 how do those non-specific cleavage happen? are they also cut by trypsin?
Thanks!
【在 l****n 的大作中提到】
: 那些peptide是non-specific cleavage引起的，这个很常见，能占到快20%，搜索的时
: 候一般都会包括这些partial cleavage的peptides。peptide的两端里有一端是non-
: specific cleavage问题不大。可信不可信主要看score， 和重复出现的次数没什么关
: 系。
|
l****n
发帖数: 711 | 7 是来自trypsin。这些酶的特异性都一般，经常认错切割位点，在不该切的地方切一刀
。其他常用的酶（chymotrypsin, GluC, ...）也一样。
【在 w********e 的大作中提到】
: how do those non-specific cleavage happen? are they also cut by trypsin?
: Thanks!
|
w********e
发帖数: 275 | 8 Thanks a lot! so seems the mass spec software knows that it can search for
fragments following amino acids that are not K and R.
usually what peptide score is trustable?
【在 l****n 的大作中提到】
: 是来自trypsin。这些酶的特异性都一般，经常认错切割位点，在不该切的地方切一刀
: 。其他常用的酶（chymotrypsin, GluC, ...）也一样。
|
fe
发帖数: 286 | 9 一般expect要0.05以下，ion score在75以上才是保险的吧，低于这个分数的不一定就
不是正确序
列，可以手工确认一下。另外还要看parent ion的delta value是不是在所用mass
analyzer的合
理误差范围内
for
【在 w********e 的大作中提到】
: Thanks a lot! so seems the mass spec software knows that it can search for
: fragments following amino acids that are not K and R.
: usually what peptide score is trustable?
|
l****n
发帖数: 711 | 10 看你用什么software了。Sequest 一般用Xcorr1.8，2.5, 3.5, deltaCN 0.08; X!
tandem 用E-value < 0.01, mascot用ion-identity>0 。在这些score以上的就非常可
信。
有的版本的软件也会直接告诉你比如95% confidence，也就是20个里面错1个。
基本上根据score判断的对错都是个统计学上的概念。一般说结果的时候都会提一下可
信度，比如"at 1% false discovery rate, 200 peptides/proteins are identified"。
【在 w********e 的大作中提到】
: Thanks a lot! so seems the mass spec software knows that it can search for
: fragments following amino acids that are not K and R.
: usually what peptide score is trustable?
|
K******S
发帖数: 10109 | 11 trypsin cuts K/R EXCEPT KP/RP and when there are acidic amino acids around
like E, you will expect miss cleavage. And trypsin usually have 1-2 miss
cleave depending on individual protocol. (You will have to try it on some
standard proteins to find out the usual miss cleavage rate)
It's normal that same peptide appears more than once (assuming from LC-
MS/MS data) because there are a lot of data points during peptide elution
time. Some instrument setting can eliminate it to a certain level. But for
high abundant and easy-to-fly peptides, you will see them multiple times.
But
【在 w********e 的大作中提到】
: I'm not familiar with protein mass spec,I'd appreciate some help for a
: question.
: The protein samples was digested by trypsin, which cuts after K and R. But
: why some peptide fragments follow amino acids other than K and R in the
: result file? and the same fragment appears more then once. are those
: fragments real?
: Thanks a lot!
|
