s*********t
发帖数: 600 | 1 SILAC可以。
传统的质谱为什么不能？比如，在A样品里面，某个蛋白的某段peptide打到36次，而在
B样品里面只打到6次，难道不能说明在A里面这个蛋白比B富集6倍吗？
多谢！ |
k******0
发帖数: 1073 | 2 短肽的电离不是定量的。
Silac是假设同一溶液中同样序列的短肽的电离效率是一样的。 |
s*********t
发帖数: 600 | 3 同一个短肽，在不同的样品里面（比如样品A和B）电离效率也不一样？
【在 k******0 的大作中提到】
: 短肽的电离不是定量的。
: Silac是假设同一溶液中同样序列的短肽的电离效率是一样的。
|
s******y
发帖数: 28562 | 4 对
电离效率和样品的制造过程有关，所以无法统一
【在 s*********t 的大作中提到】
: 同一个短肽，在不同的样品里面（比如样品A和B）电离效率也不一样？
|
s*********t
发帖数: 600 | 5 那同样的制备过程，电离效率能一样吗？
或者同一个样品，制备好，平均分成两份上样打质谱，电离一样吗，同一个peptide打
到的次数差不多吗？
【在 s******y 的大作中提到】
: 对
: 电离效率和样品的制造过程有关，所以无法统一
|
p*****1
发帖数: 330 | 6 这得分离子化方式，以及质量分析器。
【在 s*********t 的大作中提到】
: 那同样的制备过程，电离效率能一样吗？
: 或者同一个样品，制备好，平均分成两份上样打质谱，电离一样吗，同一个peptide打
: 到的次数差不多吗？
|
s*********t
发帖数: 600 | 7 那就是说质谱定量不一定非得做SILAC了。只要有一种固定得离子化方法和某些型号的
质量分析器。
【在 p*****1 的大作中提到】
: 这得分离子化方式，以及质量分析器。
|
s******y
发帖数: 28562 | 8 无法做到，因为“样品制备”包括了上样的过程，就是说，即使是同一个样品，
分开不同时间来上样，打出来的结果也不一样。
但是，质谱勉强可以用来进行半定量，比方说同一个蛋白，如果在样品一里面
出现了几千个hit,在样品二里面有十来个hit, 那么你勉强可以推测，样品一里面
含有更多的这个蛋白。
【在 s*********t 的大作中提到】
: 那同样的制备过程，电离效率能一样吗？
: 或者同一个样品，制备好，平均分成两份上样打质谱，电离一样吗，同一个peptide打
: 到的次数差不多吗？
|
c********r
发帖数: 189 | |
s*********t
发帖数: 600 | 10 2D gel灵敏度太低了
【在 c********r 的大作中提到】
: 2D gel 可以用来比较
|
|
|
m******i
发帖数: 73 | 11 ITRAQ 8plex Label
或者合成的同位素标记的标样.
对质譜来说,要绝对定量,标样是王道.
【在 s*********t 的大作中提到】
: 2D gel灵敏度太低了
|
K******S
发帖数: 10109 | 12 if your goal is quantification, you should consider MRM first. If for some
reasons you can't use MRM, you can still use spectral counting or extracted
ion chromatograms to do quantification. You just need to normalize the
sample loading and the instrument performance variation from run to run. mass
spec has been around for over 100 years. Quantitation is one of the oldest
techniques. MRM is the golden standard of small molecule quantification for
long long time.
【在 s*********t 的大作中提到】
: 那就是说质谱定量不一定非得做SILAC了。只要有一种固定得离子化方法和某些型号的
: 质量分析器。
|
l**********1
发帖数: 5204 | 13 RE LS
是吗? 王道
>同位素标记的标样.
在以下的两个单位Caltech and Scripps 里边可能只是一条叉道吧?
RE LZ: just try
Single-cell NEMS-based mass spectrometry
or Nanostructure-Initiator Mass Spectrometry (NIMS)
不需要同位素标记omics MS relative papers links:'
htp://masspec.scripps.edu/publications/public_pdf/172_greving_2011.pdf
and
htp://www.lbl.gov/tt/publications/2977pub2.pdf
or
htp://www.ncbi.nlm.nih.gov/pubmed/19514079
relative PIs web links:
http://nano.caltech.edu/people/roukes-m.html
and
htp://masspec.scripps.edu/personnel/director.php
or
htp://www.helsinki.fi/cdr/research/group_ketola.htm |
l**********1
发帖数: 5204 | 14 RE LS:
同意. 补一篇2010 Nature Reviews Molecular Cell Biology
htp://www.nature.com/nrm/journal/v11/n11/full/nrm2973.html
full text PDF link:
htp://www.imsb.ethz.ch/researchgroup/wbernd/Downloads/proteome_maps
its pp6 Fig. 1 noted this MRM proteomics assay technology...
extracted
mass
for
【在 K******S 的大作中提到】
: if your goal is quantification, you should consider MRM first. If for some
: reasons you can't use MRM, you can still use spectral counting or extracted
: ion chromatograms to do quantification. You just need to normalize the
: sample loading and the instrument performance variation from run to run. mass
: spec has been around for over 100 years. Quantitation is one of the oldest
: techniques. MRM is the golden standard of small molecule quantification for
: long long time.
|
K******S
发帖数: 10109 | 15 I think you are HU YOUed and need to read more articles.
The whole proteomics field is moving toward MRM. As I was mentioned
previously, (follow the money), just check out NCI's CPTC concept. MRM is
still the golden standard. The only reasons peptide/protein MRM is lagging
behind are techinical issues and the cost to make stable isotope labeled
standards.
Label free is not sensitive. for something with a change below 50%, you
cannot even call it a change because of the variation of the whole
technology. If you are looking for big changes, it's working fine though.
Same issue for iTRAQ, that's why you see less and less iTRAQ applications.
【在 l**********1 的大作中提到】
: RE LS
: 是吗? 王道
: >同位素标记的标样.
: 在以下的两个单位Caltech and Scripps 里边可能只是一条叉道吧?
: RE LZ: just try
: Single-cell NEMS-based mass spectrometry
: or Nanostructure-Initiator Mass Spectrometry (NIMS)
: 不需要同位素标记omics MS relative papers links:'
: htp://masspec.scripps.edu/publications/public_pdf/172_greving_2011.pdf
: and
|
K******S
发帖数: 10109 | 16 It's funny that you quoted an article from Ruedi Aebersold. Aebersold lab is
one the top labs for MRM.
【在 l**********1 的大作中提到】
: RE LS:
: 同意. 补一篇2010 Nature Reviews Molecular Cell Biology
: htp://www.nature.com/nrm/journal/v11/n11/full/nrm2973.html
: full text PDF link:
: htp://www.imsb.ethz.ch/researchgroup/wbernd/Downloads/proteome_maps
: its pp6 Fig. 1 noted this MRM proteomics assay technology...
:
: extracted
: mass
: for
|
w****s
发帖数: 235 | 17 I am curiosu which lab are you from?
【在 K******S 的大作中提到】
: I think you are HU YOUed and need to read more articles.
: The whole proteomics field is moving toward MRM. As I was mentioned
: previously, (follow the money), just check out NCI's CPTC concept. MRM is
: still the golden standard. The only reasons peptide/protein MRM is lagging
: behind are techinical issues and the cost to make stable isotope labeled
: standards.
: Label free is not sensitive. for something with a change below 50%, you
: cannot even call it a change because of the variation of the whole
: technology. If you are looking for big changes, it's working fine though.
: Same issue for iTRAQ, that's why you see less and less iTRAQ applications.
|
K******S
发帖数: 10109 | 18 small lab (my ph.d. advisor is fresh AP) but worked a lot of proteomics
projects in graduate school and still follow the field.
【在 w****s 的大作中提到】
: I am curiosu which lab are you from?
|
V***b
发帖数: 3419 | |
V***b
发帖数: 3419 | |
|
|
l****y
发帖数: 398 | 21 But MRM requires you know which protein you want to look at. It may be used
for further verification just like everyone used WB nowadays . I don't see
why MRM will dominate proteomics field.
【在 K******S 的大作中提到】
: I think you are HU YOUed and need to read more articles.
: The whole proteomics field is moving toward MRM. As I was mentioned
: previously, (follow the money), just check out NCI's CPTC concept. MRM is
: still the golden standard. The only reasons peptide/protein MRM is lagging
: behind are techinical issues and the cost to make stable isotope labeled
: standards.
: Label free is not sensitive. for something with a change below 50%, you
: cannot even call it a change because of the variation of the whole
: technology. If you are looking for big changes, it's working fine though.
: Same issue for iTRAQ, that's why you see less and less iTRAQ applications.
|
y*****x
发帖数: 14 | 22 MRM is mutiple reaction monitoring, is arguably most sensitive mass spec
analysis to monitor a known target through triple quadropole type mass
analizer
MS/MS usually means product ion scan, in protein/peptide mass spec, MS/MS
spectra were acquired to figure out the amino acid sequence in peptides,
that in most cases means to identify proteins in the sample
【在 V***b 的大作中提到】
: MRM和MS/MS有嘛区别？
|
y*****x
发帖数: 14 | 23 It might suggest there are more this protein in sample A than in sample B if
two samples are parallel prepared
【在 s*********t 的大作中提到】
: SILAC可以。
: 传统的质谱为什么不能？比如，在A样品里面，某个蛋白的某段peptide打到36次，而在
: B样品里面只打到6次，难道不能说明在A里面这个蛋白比B富集6倍吗？
: 多谢！
|
K******S
发帖数: 10109 | 24 Is this thread about "quantification" ?
I didn't say MRM will dominate the proteomics field.
used
【在 l****y 的大作中提到】
: But MRM requires you know which protein you want to look at. It may be used
: for further verification just like everyone used WB nowadays . I don't see
: why MRM will dominate proteomics field.
|
y*****s
发帖数: 1047 | 25 如果A样品的信号是36000，B样品的信号是600，基本上可以说A里面的这个蛋白比B多的
可能性很大
【在 s*********t 的大作中提到】
: SILAC可以。
: 传统的质谱为什么不能？比如，在A样品里面，某个蛋白的某段peptide打到36次，而在
: B样品里面只打到6次，难道不能说明在A里面这个蛋白比B富集6倍吗？
: 多谢！
|
y*****s
发帖数: 1047 | 26 MRM is just one type of MS/MS mostly used for quantitation |
l**********1
发帖数: 5204 | 27 So what's up next cutting edge MS proteomics method?
ZZ
Why nanoLiters?
Nanoliter Cool Wave®
exciting, inexpensive, sample handling platform & technology.
Booth 17 Improve sensitivity of MALDI, DART, DESI, SIMS, LDI 10 to 100x
LITERALLY!
Also, manipulate and spatially, temporally concentrate samples for TLC, MS,
IR, NMR, PCR; electrophoresis, fluorescence, crystallization & more.
Dispense viscous liquids non-touch (glycerol, blood, serum, ionic liqs.,
glues) for forensics, defense, medical, pharma, energy, proteomics, iomics,
apps.
Save expensive labeled stds. Use for blood spot dispensing or DNA sample
prep.
from PDF book
htp://www.asms.org/LinkClick.aspx?fileticket=FHxivrA3CSE%3D&tabid=36
at the 59th ASMS Conference on Mass Spectrometry and Allied Topics
Date: 05 Jun - 09 Jun 2011
Place: Denver, Colorado, USA
Website: http://www.asms.org
htp://www.laboratory-journal.com/event/59th-asms-conference-mass-
spectrometry-and-allied-topics
papers:
htp://www.ncbi.nlm.nih.gov/pubmed/20161086
htp://www.ncbi.nlm.nih.gov/pubmed/18479933
Ps:
60th ASMS Conference on Mass Spectrometry and Allied Topics
Vancouver, BC, Canada
May 20 - 24, 2012 • Short Courses May 19 and 20
htp://www.asms.org/Conferences/AnnualConference/GeneralInformation/tabid/127
/Default.aspx
【在 K******S 的大作中提到】
: Is this thread about "quantification" ?
: I didn't say MRM will dominate the proteomics field.
:
: used
|
b*****r
发帖数: 203 | 28 I agree.
Quantitation in mass spectrometry is always a problem. It is easy to
generate data, but it is more difficult to get reliable quantitation from
the data.
MRM is probably the easiest and relatively reliable way for quantitation.
But even for MRM, there are also chances of "measuring an ion other than the
intended target"; see the discussion on SRM in "Recommendations for mass
spectrometry data quality metrics for open access data (corollary to the
Amsterdam principles)."
Proteomics. 2011 Nov 8. doi: 10.1002/pmic.201100562. [Epub ahead of print]
J Proteome Res. 2011 Dec 8. [Epub ahead of print]
Mol Cell Proteomics. 2011 Dec;10(12):O111.015446. Epub 2011 Nov 3.
Another way is Aqua
http://www.sigmaaldrich.com/life-science/proteomics/mass-spectr but it may be expensive and/or not as convenient as MRM.
【在 K******S 的大作中提到】
: I think you are HU YOUed and need to read more articles.
: The whole proteomics field is moving toward MRM. As I was mentioned
: previously, (follow the money), just check out NCI's CPTC concept. MRM is
: still the golden standard. The only reasons peptide/protein MRM is lagging
: behind are techinical issues and the cost to make stable isotope labeled
: standards.
: Label free is not sensitive. for something with a change below 50%, you
: cannot even call it a change because of the variation of the whole
: technology. If you are looking for big changes, it's working fine though.
: Same issue for iTRAQ, that's why you see less and less iTRAQ applications.
|
b*******g
发帖数: 1309 | 29 for absolute quantitation purpose
MRM 里的标样最好也是isotope标记standard，就跟AQUA的出发点一样
所谓的AQUA 在复杂样品中根本就不行，还得上MRM，这样才能降低background 的
inteference
the
【在 b*****r 的大作中提到】
: I agree.
: Quantitation in mass spectrometry is always a problem. It is easy to
: generate data, but it is more difficult to get reliable quantitation from
: the data.
: MRM is probably the easiest and relatively reliable way for quantitation.
: But even for MRM, there are also chances of "measuring an ion other than the
: intended target"; see the discussion on SRM in "Recommendations for mass
: spectrometry data quality metrics for open access data (corollary to the
: Amsterdam principles)."
: Proteomics. 2011 Nov 8. doi: 10.1002/pmic.201100562. [Epub ahead of print]
|
K******S
发帖数: 10109 | 30 what the heck is this?
,
【在 l**********1 的大作中提到】
: So what's up next cutting edge MS proteomics method?
: ZZ
: Why nanoLiters?
: Nanoliter Cool Wave®
: exciting, inexpensive, sample handling platform & technology.
: Booth 17 Improve sensitivity of MALDI, DART, DESI, SIMS, LDI 10 to 100x
: LITERALLY!
: Also, manipulate and spatially, temporally concentrate samples for TLC, MS,
: IR, NMR, PCR; electrophoresis, fluorescence, crystallization & more.
: Dispense viscous liquids non-touch (glycerol, blood, serum, ionic liqs.,
|
|
|
b*****r
发帖数: 203 | 31 你说的很对。
【在 b*******g 的大作中提到】
: for absolute quantitation purpose
: MRM 里的标样最好也是isotope标记standard，就跟AQUA的出发点一样
: 所谓的AQUA 在复杂样品中根本就不行，还得上MRM，这样才能降低background 的
: inteference
:
: the
|
b*****r
发帖数: 203 | 32 下面的review paper提到各种定量方法
Current trends in quantitative proteomics.
Elliott MH, Smith DS, Parker CE, Borchers C.
J Mass Spectrom. 2009 Dec;44(12):1637-60. Review.
【在 s*********t 的大作中提到】
: SILAC可以。
: 传统的质谱为什么不能？比如，在A样品里面，某个蛋白的某段peptide打到36次，而在
: B样品里面只打到6次，难道不能说明在A里面这个蛋白比B富集6倍吗？
: 多谢！
|
t******t
发帖数: 3045 | 33 AQUA不用ms/ms？full scan的snr和specificity太差了点 |
l**********1
发帖数: 5204 | 34 RE
单一个分离column进样量从 0.5 ul(500nl) vs 20nl 的质谱
量变就可能成为质变啦, sn线性/回归性上
更何况人家US+瑞士的实验室在开发96/394 分离column 同时进样量2 nl 的
Matrix Nanoliters MS吧?
htp://www.imsb.ethz.ch/researchgroup/wbernd
Our nanostructure-based mass spectrometry platform, a novel matrix-free surface analysis technology, is being developed for metabolite tissue imaging, activity screening and analyzing nano-arrays.
htp://masspec.scripps.edu/personnel/director.php
htp://www.scs.illinois.edu/sweedler/publications.html
【在 K******S 的大作中提到】
: what the heck is this?
:
: ,
|
l**********1
发帖数: 5204 | 35 Did you or your boss attended below conference?
59th ASMS Conference on Mass Spectrometry and Allied Topics
Date: 05 Jun - 09 Jun 2011
Place: Denver, Colorado, USA
Website: http://www.asms.org
htp://www.laboratory-journal.com/event/59th-asms-conference-mass-
spectrometry-and-allied-topics
or plan to attend next round:'
60th ASMS Conference on Mass Spectrometry and Allied Topics
Vancouver, BC, Canada
May 20 - 24, 2012 • Short Courses May 19 and 20
htp://www.asms.org/Conferences/AnnualConference/GeneralInformation/tabid/127/Default.aspx
or Pittcon 2012
Course Information
Course Title: How To Launch 100% of Liquid Samples Into ESI Mass Spectrometers and More About the Nanoliter Regime
Categories: 1 - Liquid Chromatography-Mass Spectrometry
2 - Mass Spectrometry
3 - Proteomics
4 - Sample Preparation
htp://www.nanoliter.com/newsevents.htm
【在 K******S 的大作中提到】
: small lab (my ph.d. advisor is fresh AP) but worked a lot of proteomics
: projects in graduate school and still follow the field.
|
l**********1
发帖数: 5204 | 36 RE:
纳升级的和微升级的omics nr和specificity 能一样吗?
pls efer
34F nanolitersMSdemo figure shown
【在 t******t 的大作中提到】
: AQUA不用ms/ms？full scan的snr和specificity太差了点
|
K******S
发帖数: 10109 | 37 are you a sales person from those places? hehe
【在 l**********1 的大作中提到】
: Did you or your boss attended below conference?
: 59th ASMS Conference on Mass Spectrometry and Allied Topics
: Date: 05 Jun - 09 Jun 2011
: Place: Denver, Colorado, USA
: Website: http://www.asms.org
: htp://www.laboratory-journal.com/event/59th-asms-conference-mass-
: spectrometry-and-allied-topics
: or plan to attend next round:'
: 60th ASMS Conference on Mass Spectrometry and Allied Topics
: Vancouver, BC, Canada
|
l**********1
发帖数: 5204 | 38 不是，我目前不做质谱了
顺便问下
问你或你老板去/打算去 2011/2012质谱年会没有
http://www.asms.org/Conferences/AnnualConference/GeneralInforma
和我是那个技术的公司推销员 有啥关系?
你/or你老板不见得从不参加质谱年会?
【在 K******S 的大作中提到】
: are you a sales person from those places? hehe
|
