i have collectd proteins and peptides from size exclusion chromatography and then performed MALDI-MS. in results there are lots of peaks with different masses how can i characterize or identify these proteins/peptides masses which are undigested/ intact.
Dear Atif,
maybe you can provide some additional information, e.g. in which mass range your peptides /proteins are? Which matrix was used? Which mass analyzer did you use (TOF?, Orbitrap?, others?).
For proteins, in some cases not only the singly charged ions are observed in MALDI MS but you can also see the doubly/triply or even more charged species. Maybe this contributes to the high number of signals. Other possibilities are adducts (e.g. Na+, K+, NH4+ etc) but these are only only resolved in case of smaller proteins; for bigger proteins they contribute certainly to broad signals.
Identifcation of intact proteins by MALDI MS is - in principle - possible for smaller proteins, however, I would clearly recommend digestion. This can also be performed directly on the target.
best regards
andreas
Yes, I am agree with professor Andreas Tholey.
1. First decided which mass range of the protein and peptide looking for.
2. Also which matrix using for protein and peptides identification. You can used HCCA (Peptides and proteins ), SA (Proteins) and 2,5-DHB (Proteins and Oligosaccharides). You can select according to the result best choice of the matrix for identify proteins/peptides masses in your desire collected sample from size exclusion chromatography.
If looking for smaller protein, I will recommend you for digestion.
Regards
Aisha
What is your initial sample? How many proteins do you have in it ?
Dear Atif,
Maybe I am saying something obvious but you should digest your proteins and run mascot (or analog software) (http://www.matrixscience.com/cgi/search_form.pl?FORMVER=2&SEARCH=PMF) with the list of peptides masses. Unless you already know or have an idea of which proteins you should have is quite difficult to identify the protein from just the protein mass. Also you have to consider post-translational modifications.
Best
Matteo
Hello All and thank you for your kind response.
Dear Andreas Tholey,
we are using MALDI-TOF TOFultra flex III software flex analyzer compass 1.3 and matrix Sinapinic Acid and DHB in positive mode.
Matteo Scalabrin,
i have used tryptic digested peptides on MALDI and match peptides masses on mascot to match peptides but i want to perform intact protein (GFC eluted peak which has mass range 50kDa to 75kDa).
Keerthi Bandara Jayasundera,
exactly not confirm how many proteins are in this GFC fraction but this GFC fraction shows two bands on SDS-PAGE.
i actually want to see the MALDI spectra pattern of my protein, Masses of intact proteins and dimer, tetramer n oligomer of these proteins. is it possible to identify intact protein and its property with the help of MALDI?
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