tryptic digestion of ovalbumin

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msonline
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tryptic digestion of ovalbumin

Postby msonline » Wed Jan 16, 2013 6:50 pm

Hi, I am starting an expt. on protein identification by mass spectrometry. I have used chicken ovalbumin as my model system. After in solution tryptic digestion, the digested ovalbumin solution was purified through C18 ZipTip (Millipore) as manufactor's instruction. After elution step, the sample was disolved in 50% MeOH with 1% acetic acid, and was then directly loaded on a nanospray tip for nanoESI-MS.
Here comes the results: I have assigned some peaks to ovalbumin peptides. However, around m/z500-900, the intensity of peaks distribute like a polymer, with mass difference 14 Da. (Seems to be polyethylene?) I have conducted same expt. on bovine serum albumin, and no polymer-like peak distribution was observed. I am really confused with the polymer-like peak distribution in digested ovalbumin, and what to find out where it comes from? and how to remove that 'contamination'? Does anyone have the same experience? or give me some suggestion? Thanks.

PS. Ovalbumin was bought from Sigma, and was lyophilized powder with purity ≥98% (agarose gel electrophoresis). It is a glycoprotein containing only single site of N-glycosylation.

karthikskamath
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Postby karthikskamath » Sun Jan 20, 2013 8:04 pm

In case you have doubts about having polymers, there are many chances how they are introduced into your sample. First simple check is compare the sample with blank solvent run and check for the presence of similar patters in blank solvent. Technically your tryptic digested peptide should not show a huge charge distribution envelope (if it is still undigested yes it might show). Hence check into isotope distribution of those peaks for knowing what they really are. Also take the sequence of the proteins and perform a insilico-digest over MS Digest. This should give you idea about masses of tryptic peptides you must expect. Beyond this if you could give your e-mail ID, I can post you two PDFs which has list of common contaminants. You can compare the masses of those peaks with that of list. Good luck..

msonline
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Postby msonline » Tue Jan 22, 2013 7:17 am

My email: felixwang08@gmail.com Really appreciated if you send me that PDFs. Performing a blank solvent run is good, but i still see some polymer peaks. This time it shows a mass difference of 82 Da. Until now, I have experienced three different polymer envelops with different mass difference (14, 44, and 82 Da) in three experiments. I guess maybe chemical reagents used contain polymer impurities. Maybe the C18 desalting process purifies these polymers as well as targeted peptides.

Thank you so much.


karthikskamath wrote:In case you have doubts about having polymers, there are many chances how they are introduced into your sample. First simple check is compare the sample with blank solvent run and check for the presence of similar patters in blank solvent. Technically your tryptic digested peptide should not show a huge charge distribution envelope (if it is still undigested yes it might show). Hence check into isotope distribution of those peaks for knowing what they really are. Also take the sequence of the proteins and perform a insilico-digest over MS Digest. This should give you idea about masses of tryptic peptides you must expect. Beyond this if you could give your e-mail ID, I can post you two PDFs which has list of common contaminants. You can compare the masses of those peaks with that of list. Good luck..

karthikskamath
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Postby karthikskamath » Tue Jan 22, 2013 8:30 pm

The reasons for introduction of such polymers starts with simplest of things such as tubes and tips you use in the process. I did face similar problem quite few times. According to my experience 44Da difference peaks correspond to Polyethylene glycol. I would suggest to deconvolute the spectrum look at the mass of the peak with that of list which I have provided. The list provides the possible identity of the polymers and possible source of identity and ways to avoid the same. These polymers competitively ionize better and hence appear as major peaks. Good luck.

msonline
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Postby msonline » Thu Jan 24, 2013 5:41 am

Thank you for the suggestion. I increased my sample concentration, and now tryptic digested peptides peaks became major peaks. Although polymer peaks still exist, they are negligible.


karthikskamath wrote:The reasons for introduction of such polymers starts with simplest of things such as tubes and tips you use in the process. I did face similar problem quite few times. According to my experience 44Da difference peaks correspond to Polyethylene glycol. I would suggest to deconvolute the spectrum look at the mass of the peak with that of list which I have provided. The list provides the possible identity of the polymers and possible source of identity and ways to avoid the same. These polymers competitively ionize better and hence appear as major peaks. Good luck.


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