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A series of peaks in deconvoluted result

Posted: Sat Jun 10, 2017 8:38 pm
by davidfan05
I am pretty new to MS field. I just started a transferrin project. I am trying to develop a method for semi-quatify the ratio of mono-glycosylated transferrin/transferrin. I am using Agilent 6550 iFunnel Q-TOF LC/MS. Right now, I am trying with normal transferrin, i.e. di-glycosylated transferrin (about 79 kD). The UPLC is Agilent 1290 Infinity II. I tried two columns, and get a deconvoluted result showing a series of peaks with 240 D difference in mass. I do not know what this means. Does this mean that transferrin combine with a chemical of 240 D and form an aggregate with different number of chemical molecules? How can I solve this issue and get a typical peak? See detailed results at: https://drive.google.com/file/d/0BxVutI ... sp=sharing

Thank you for your attention and help!

David

Re: A series of peaks in deconvoluted result

Posted: Sun Jun 11, 2017 7:59 am
by Rndirk
I'm not into protein LC-MS, but I remember from my studies that this is typical. The m/z differences you see are -correct me if i'm wrong- losses of amino acids in combination with differences in charge state . This can be used for protein identification.

You should probably dig into the theory of doing protein analysis with QTOF.

Aside from that, i think your chromatogram shows that you're overloading the column (concentration too high).

Re: A series of peaks in deconvoluted result

Posted: Sun Jun 11, 2017 9:20 pm
by davidfan05
Thank you, Rndirk. You are right. The figure that I showed suggested that I overloaded the sample. I checked the result of lower concentration, and it was much better in term of bump issue. However, there is still issue of multiple peaks. What are these peaks? How can I suppress other peaks? Thanks!

Re: A series of peaks in deconvoluted result

Posted: Fri Jun 16, 2017 3:18 pm
by davidfan05
Is this because of the contamination of detergent? We did have a chance to be contaminated by NP-40.

Re: A series of peaks in deconvoluted result

Posted: Sun Jun 18, 2017 2:36 am
by JMB
What is the mobile phase ?
What is the sample dissolved in ?

Regards,
JMB

Re: A series of peaks in deconvoluted result

Posted: Sun Jun 18, 2017 2:37 am
by JMB
What is the mobile phase ?
What is the sample dissolved in ?
What is the scan range ?

Regards,
JMB

Re: A series of peaks in deconvoluted result

Posted: Mon Jun 19, 2017 11:19 pm
by davidfan05
The mobile A phase is 0.1% formic acid in H2O, and the mobile B phase is 0.1% formic acid in ACN. The sample is dissolved in MS quality water, and the scan range is 74 kDa to 81.5 KDa.

Right now, I kind of figure out that this has something to do with the water I used to prepare the samples for loading. When I managed to load the sample freshly, I will get one peak. However, 30 min later, when I re-measured this sample, it showed more peaks. I suspect that my protein is interacting with the extracts from container plastic or glass. How could I solve this problem?