g****e
发帖数: 137 | 1 the most classic way to do this is to use radioactive SAM or stable isotope
labeled methyl group donor. There are many pan-methylation antibody
available, but don't expect too much for them. Unlike acetylation and many
other PTM, methylation has mono, di and tri-methylation, that's why is
difficult to develop an antibody recognize all three types of methylation. |
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m*******n
发帖数: 387 | 2 谢谢回复
那要是把这个蛋白纯化出来，做质谱如何？
isotope |
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g****e
发帖数: 137 | 3
Yes, it's doable but it's not as sensitive as isotope labeled protein. |
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K******S
发帖数: 10109 | 4 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
te... 阅读全帖 |
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b*******g
发帖数: 1309 | 5 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 |
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l**********1
发帖数: 5204 | 6 Did you try test the level of leakiness with no isotope label actin?
i mean whether your target even abundantly but still holded entirely intra- cellular tyrosine-
containing protein, such as actin or tubulin:
then your negative control with 35S labeled that also became Immunoprecipitation positively
Details please go to
Protocol full text link:
//webdoc.nyumc.org/nyumc/files/sun-lab/attachments/CPCB.ch07.Protein%
20Labeling.pdf
cited :
>For many applications, an important control for surface-l... 阅读全帖 |
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r***n
发帖数: 52 | 7 Thanks for your help. Actually, I'm not using isotope to label protein.It's
isotype for antibody control. Because all of papers published with IP
results showed IgG control, which is supposed to be no band of target. But
mine has the same band intensity as IP ones but wash off some after long
time incubation with TBST and detected by another species HRP.
- cellular tyrosine-
Immunoprecipitation positively
experiments |
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l**********1
发帖数: 5204 | 8 Did you try test the level of leakiness with no isotope label actin?
i mean whether your target even abundantly but still holded entirely intra- cellular tyrosine-
containing protein, such as actin or tubulin:
then your negative control with 35S labeled that also became Immunoprecipitation positively
Details please go to
Protocol full text link:
//webdoc.nyumc.org/nyumc/files/sun-lab/attachments/CPCB.ch07.Protein%
20Labeling.pdf
cited :
>For many applications, an important control for surface-l... 阅读全帖 |
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r***n
发帖数: 52 | 9 Thanks for your help. Actually, I'm not using isotope to label protein.It's
isotype for antibody control. Because all of papers published with IP
results showed IgG control, which is supposed to be no band of target. But
mine has the same band intensity as IP ones but wash off some after long
time incubation with TBST and detected by another species HRP.
- cellular tyrosine-
Immunoprecipitation positively
experiments |
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l**********1
发帖数: 5204 | 10 SILAC Stable isotope Mass Spectrometry Analysis is more general than that Cell paper used special antibody
labeled for quantitative analysis in proteomics.
|
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l****y
发帖数: 398 | 11 the best way is metabolic isotopic labeling, then quantitative mass spec. |
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a*****x
发帖数: 901 | 12 Just my two cents, not in the field...
1) pulse chase (fluore or isotope) or CHX treatment or SNAP tag with or
without over-expression of E (or knock-down)
2) Phospho versus dephospho mimic mutation; If the sites are not clear, try
to look for phospho change by over-expression of E (or knock-down)
3) specific inhibitors |
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g*********5
发帖数: 2533 | 13 1.大量磷酸化motif..
你先比较下同源蛋白，做个clustalw
先研究保守的。
2.L truncate form，看那一段和E结合。磷酸化位点应该在结合的那段上
pulse chase (fluore or isotope)， fluore如何做？谢谢。 |
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l**********1
发帖数: 5204 | 14 SILAC (Stable isotopic atoms for quantitative
mass spectrometry analysis)
Plus spatial proteomics eGFP/alternative biomarker labelling
details
please go to
original paper link:
//www.ncbi.nlm.nih.gov/pubmed/21937730
or
2011 one review:
//www.ncbi.nlm.nih.gov/pubmed/21476986
To perform a system-wide analysis of
protein turnover
In human proteome:
cultured human cells. Protein abundance and
the rates of protein synthesis, degradation and turnover have
been measured in parallel for whole cells and... 阅读全帖 |
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g*********5
发帖数: 2533 | 15 this one is strong than 32p? |
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l**********1
发帖数: 5204 | 16 还好 没扔在32^P isotope lab area bin |
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b*******g
发帖数: 1309 | 17 ITRAQ方便，protein digestion 之后开始标记，也相对便宜
SILAC 从cell culture开始加isotope labeled AA
这个方法对in vivo的实验没办法
ionization efficiency for some iTRAQ labeled peptides might be enhanced,
this would boost the sensitivity.
linear range for quantitation, SILAC might be better. |
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y*****s
发帖数: 1047 | 18 Not considering matrix effect, current LC-MS are reproducible and linear,
owing to robustness and precision of current instrumentation
If you inject the same sample multiple times, CV should be <20% if SNR is >5
And you should easily get a straight line through standard addition
However, mass spec is not a spectroscopic detection method by nature
sample matrix greatly impacts the signals,
1 pmol/L of peptide X in 10% acetonitrile shows a signal of 100, but the
same level of peptide in a plasma d... 阅读全帖 |
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r******t
发帖数: 135 | 19 Thanks for suggestions. GE Typhoon 9400 is very expensive, and the function
for detecting isotope is not a plus for us. |
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r******t
发帖数: 135 | 20 Thanks for suggestions. GE Typhoon 9400 is very expensive, and the function
for detecting isotope is not a plus for us. |
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K******S
发帖数: 10109 | 21 TIC is meaningless. You can probably ignore your 1st figure.
2nd figure (sounds like MS1), you need to zoom in the area where you detect
the intact peptides, for high resolution data, you need to show all the
isotopic peaks and at least 4 decimals of m/z, such as 1000.0000 (assuming
you use high resolution instrument)
3rd figure you mentioned sounds like MS2 spectra, did you label the
fragments? |
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l**********1
发帖数: 5204 | 22 if that PCR mutation coincidencely happend on the key ponit or region of the
binding sites between that
probe and Genomic DNA sample, which is simialr to loss of docking site in
Western Blotting , then how can you expect both bind tightly unless p32 or
s35 etc isotope labelling can pick weak binging up but Dig labelling Can't
pick that kind of weak binging.
And after sub cloning that the slected white clone can keep your probe
almost no changed and inserted into that vector with LacZ gene of β-
... 阅读全帖 |
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r***e
发帖数: 2539 | 23 要不用roche的那个AP标记，做southern?
灵敏度比isotope略差，做genotyping应该够了，至少比较安全。 |
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l**********1
发帖数: 5204 | 24 Dozen Ys ago,
auto sequencer ABI Prism 3100 or 3700
15 Ys ago ABI 310 or 373 already didn't use Sanger isotope labelling..
Pls refer,
http://www.ncbi.nlm.nih.gov/pubmed/10220426
Ps:
matrix used almost above types then.. |
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l**********1
发帖数: 5204 | 25 Dozen Ys ago,
auto sequencer ABI Prism 3100 or 3700
15 Ys ago ABI 310 or 373 already didn't use Sanger isotope labelling..
Pls refer,
http://www.ncbi.nlm.nih.gov/pubmed/10220426
Ps:
matrix used almost above types then.. |
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g*********s
发帖数: 194 | 26 Boss: Shawn Burgess
Biological NMR and MS
In Vivo and Ex Vivo isotope tracer techniques
Intermediary metabolism of obesity and diabetes
Regulation of carbohydrate and lipid metabolism in liver
最好fresh Ph.D |
|
L****r
发帖数: 333 | 27 如果你想知道这些碎片是什么 （通常我们说的定性），你得用UPLC/HPLC-TOFMS或者
UPLC/HPLC-Orbitrap，如果有FTICR更好，这样你就能得到他们的isotopic mass （通
常我们称作精确分子量）, 注意，离子峰里面有很多是multiple-charge,你得去分析他
们， 为什么用柱子，前面的已经提到过，防止离子抑制，就是说，很多不同种类的中
性分子一道出来，在离子化的时候，有些离子被抑制掉了，这样你就得不到他们的峰，
用HPLC，他们就会一个一个地离子化，大大降低他们被抑制的可能。
然后定量，使用UPLC/HPLC-TRIPLEQUAD MS， 使用MRM，即用他们的碎片定量，一旦确
定是什么PEPTIDE，碎片就很容易得出，即使两个PEPTIDES有相同的分子量，不同结构
和相同的RETENTION TIME， 该仪器也能准确地定量他们。 |
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i****g
发帖数: 3896 | 28 不知道你为什么这么喜欢臆想一些毫无根据的可能，说得越多越当真似的。
Kobilka在Nature发b2AR结构的几乎同时，Raymond Stevens也和他在Science发了两篇
back to back的b2AR结构文章，两人都为通讯作者。之后Stevens又单独或和其他人发
了几篇。Stevens是结构生物学家，以前不做GPCR，也和施一公一样研究很多蛋白结构
，发了不少CNS。但最后是Kobilka和导师Lefkowitz得了炸药奖，Stevens没得。而
Lefkowitz的工作从来就和b2AR的晶体结构无关。
这是Kobilka和Lefkowitz获诺贝尔奖的原因，30-40年的工作，b2AR结构只是GPCR三个重
大发现中的一个：
http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2012
"for studies of G-protein–coupled receptors"
Lefkowitz started to use radioactivity in 1968 in order to trac... 阅读全帖 |
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K******S
发帖数: 10109 | 29 you need stable isotope labeled satndard peptide and LCMRM, routine/typical
mass spec quant workflow. |
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l*******t
发帖数: 1016 | 30 美国华人生物医学发考题猝死现象
刚来美国做博后就听说个一个华人biomedical faculty 手里有三个R01，有一次做
presentation讲到激动处昏厥 然后就挂了。 还有一次有个华人faculty candidate 来
我们学校面试presentation 不到40岁 可是已经满头白发看起来非常苍老。华人Biomed
千老有死于胃癌的 女千老怀毛毛下面就流血的等等。这可能有以下原因：
1）工作时间长 三点一线 》60h/w
2）跑胶克隆核酸纯化也是辛苦体力活
3）读写说查都用英文 费脑力
4）大量杀生（杀老鼠等）造业很大
5）使用isotopes, carcingoens, HIV FIV lentivirus 等使免疫力下降
6) 没法在家上班 风雨雪通勤开车也紧张
7）和国内同学比 当不上发考题没有面子 压力大
8）NIH funding竞争性太强
9）实验室内多是华人 处理关系费事
10）多数没有信仰 不能meditate或者太极放松
建议搞生物医学的发考题和千老，最好学学佛教 会有很大的收获和人生观的改变。念
金刚经会使脑筋得到极大休息 念地藏经会消除杀老鼠的... 阅读全帖 |
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l****y
发帖数: 398 | 31 不行，有isotope envelope, 至少得差4 Da才行。 |
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p******n
发帖数: 55 | 32 http://www.grc.org/amc.aspx
熟悉的名字不少啊
2016 Lecturers
◾Nieng Yan, Membrane Transport Proteins
◾Xiaowei Zhuang, Synaptic Transmission
◾Christopher J. Chang, Metals in Medicine
◾Mikhail Lukin, Quantum Science
2015 Lecturers
◾Karl Deisseroth, Amygdala in Health & Disease
◾David M. Sabatini, Lysosomal Diseases
◾Laura Kiessling, Carbohydrates
◾Gerbrand Ceder, Physical Metallurgy
◾Liang Fu, Superconductivity
2014 Lecturers
◾Kristi S. Anseth, Si... 阅读全帖 |
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g*****x
发帖数: 3283 | 33 定量proteomics一般是用stable isotope标记的样品做internal reference，这个我没
亲自做过不敢乱说
: 还有想抓住这个难得的机会请教一下MS业内人士，我想对比NGS和MS来进一步理
解一下
: MS。我做NGS比较所多，MS只做过两个项目，在做第三个，但是都比较肤浅，毕
竟上游
: 的分析不是我们自己做。NGS上下游都自己做，所以理解相对多一些。
: 对于RNA-seq，total RNA样本，和MS cell lysate对应，都是复杂样品，测出来
的序列
: 数目和测序深度（花钱多少）正相关。一般样本，比如说人的细胞total RNA，
mRNA-
: seq，需要100 ng total RNA来建库，测100 Million以上的short reads基本上
就到平
: 台了，一个lane现在可能就HiSeq 4000一千刀左右或者更便宜。而且可以定量每
个gene
: 的表达量或者每个isoform的表达量。
: 那么对于Mass Spec，同样的人的细胞的cell... 阅读全帖 |
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i*********w
发帖数: 134 | 34 俺感觉没这么严重把，替作者辩护辩护
1) Undefined characterizations for MTT. Poor proton NMR, no 13C NMR, no Mass
spectrum.
reply: MTT合成是报道过的，H NMR确认一下可以了
2) Poor ESI-MS for melamine. The signals are broad (like inorganic species),
no isotope distribution and no fragmentation species, inadiquate to claim
the identity of the analyte.
reply:可能是仪器的问题吧。有没加melanmine的牛奶作对比，俺觉得可以接受
3) Postadded images possibly selective or made-up. Also some images are
inconsistent (Compared these images without melamine added, 7.5 ppb a |
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b******3
发帖数: 62 | 35 Your help is greatly appreciated.
Applied Radiation and Isotopes
Volume 52, Issue 1, January 2000, Pages 55-61 |
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w********g
发帖数: 447 | 36 从你的mass来看，唯一的两个可能就是
C8H12O4和C4H8N6O2。
如果看fragmentation，所有的fragment按照N规则，似乎都是不含N的，要所有的
fragment都不含N，那么分子本身含N的可能性比较小。也就是说第二个的可能性比较小
。如果你的isotopic peak的ratio是准的话，和第二个分子式match，这样又排除了第
一个。而且第二个分子式算出来的mass和你测得的只差1.7个ppm，对于FTMS来说比较
make sense。而第一个分子式的计算值和你测得要差17.3个ppm，对于FTMS来说有点太
大了。
感觉分子式C4H8N6O2，但是回到开始说的N规则，如果有6个N的话，你所有的
fragmentation的碎片里面either要含有所有的6个N，或者每次掉偶数个N。另外看你的
fragmentation，碎片几乎都分布在比较靠近molecular ion的一端，低mass几乎看不到
太多的fragmentation，感觉最大的可能是你的6个N都不太容易掉，比如都长在几个共
轭的环上，环上有些支链。但你又说是个直链的东西，那可能是一些直链 |
|
h*****7
发帖数: 227 | 37 从第二张谱图上的标题看，是ion trap。
大家觉得在MS/MS里，isotope的信息的作用有多少，个人感觉受很多因素的影响，很多
化合物的结果不太一致。 |
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b*******g
发帖数: 1309 | 38 我就是做MS的，所以估计不用你来教育我MS的知识
至少我看来，MS数据不严谨，但是你也不可能从一个不严谨的数据得到不是melamine的
结论？
你要完全确认这个结构至少要高分辨MS，加MS/MS，否则你不能说不是melamine
Malamine 的第一个isotope峰只有5%，如果是分辨率不好的ESI，确实就是这样的峰
另外你就不用教育我了，没有确凿证据，你就是在这里喊一万遍，也没屁用，
我是周末闲着没事才无聊回复你的。
Major |
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t******t
发帖数: 3045 | 39 15N is a stable isotope |
|
|
|
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q****i
发帖数: 6923 | 43 isotec,
cambridge isotope laboratories |
|
u**s
发帖数: 384 | 44 【作者】Weixun Wang,† Haihong Zhou,† Hua Lin, Sushmita Roy,
Thomas A. Shaler, Lander R. Hill, Scott Norton, Praveen Kumar, Markus
Anderle, and Christopher H. Becker*
【文题】Quantification of Proteins and Metabolites by Mass Spectrometry
without Isotopic Labeling or Spiked Standards
【期刊名，年份，卷（期），起止页码】Anal. Chem., 2003, 75 (18), pp 4818–
4826
【全文链接】http://pubs.acs.org/doi/pdfplus/10.1021/ac026468x
【求助者联系方式】b*********[email protected] |
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b*******g
发帖数: 1309 | 45 你说的应该是M 1+ 跟 M2+ 峰
看isotope 的 间距， 差1Da的就是M 1+
差0.5 Da 的就是M2+ |
|
|
f*****t
发帖数: 2 | 47 今天在 itune 上偶然发现一个应用程序 chem helper,
http://itunes.apple.com/us/app/chem-helper/id416904372
其有如下功能:
Chemical Equation Balancer
Molecular Weight Calculator
Periodic Table
NMR Frequency Calculator
Stoichiometry
Buffer Calculator
Isotopic Distribution
我主要是想问问, 它的这些功能里哪些是最常用的? 如果都很有用的话，就准备给儿子
买一个。请帮忙看一下，谢过了。 |
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f*****t
发帖数: 2 | 48 谢谢回复。那个 Isotopic Distribution 是不是 too advanced for highschooler 呀
? |
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W*******e
发帖数: 817 | 49 Stable isotope labelling and FPLC–ICP-SFMS for the accurate determination
of clinical iron status parameters in human serum
M. Estela del Castillo Busto, Maria Montes-Bayón, Jörg Bettmer and
Alfredo Sanz-Medel
Analyst, 2008, 133, 379-384 |
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h*****t
发帖数: 1226 | 50 many methods, for exapmple, isotope labeling,
but I think what you heard might be MRM quantitation.
量测 |
|
