A series of peaks in deconvoluted result

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

9 posts Page 1 of 1
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
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).
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!
Is this because of the contamination of detergent? We did have a chance to be contaminated by NP-40.
What is the mobile phase ?
What is the sample dissolved in ?

Regards,
JMB
What is the mobile phase ?
What is the sample dissolved in ?
What is the scan range ?

Regards,
JMB
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?
By changing the gradient and other parameters, I am now able to get mainly one peak. However, it seems that I need to make the sample freshly. With time, somehow I will get multiple peaks again. Also with time, it seems less likely to get good multiple charge envelops in Spectrum result.

What is the cause of this issue? How can I solve this problem?

Thank you for your attention and help.

D
What was the initial m/z range that was scanned, not the de convoluted MW RANGE ?

Can you post a spectrum, not deconvoluted, so that we can possibly get a fix on the adduct ions.
Once those are identified, it may be possible to know their source.
9 posts Page 1 of 1

Who is online

In total there is 1 user online :: 0 registered, 0 hidden and 1 guest (based on users active over the past 5 minutes)
Most users ever online was 1117 on Mon Jan 31, 2022 2:50 pm

Users browsing this forum: No registered users and 1 guest

Latest Blog Posts from Separation Science

Separation Science offers free learning from the experts covering methods, applications, webinars, eSeminars, videos, tutorials for users of liquid chromatography, gas chromatography, mass spectrometry, sample preparation and related analytical techniques.

Subscribe to our eNewsletter with daily, weekly or monthly updates: Food & Beverage, Environmental, (Bio)Pharmaceutical, Bioclinical, Liquid Chromatography, Gas Chromatography and Mass Spectrometry.

Liquid Chromatography

Gas Chromatography

Mass Spectrometry