b*****y
发帖数: 175 | 1 My protein was eluted in 0.3% vitamin C in H2O and lyophilized (vitamin C
was intended to prevent any Met oxidation of my protein). Since I know
highly acidic pH is not good for LC-MS so I tried three different approaches:
1. directly dissolve in ammonium bicarbonate and adjust pH to 8
2. directly dissolve in ammonium bicarbonate and dialyzed against ammonium
bicarbonate (3 changes in 6 hrs) and the final pH is 8
3. dissolve in ammonium bicarbonate and buffer exchanged to 3M guanidinium
hydrochloride in Tris buffer and dialyzed against ammonium bicarbonate (3
changed in 24 hrs). This approach should unfolds the protein at first and
let it refolds.
Then I went to perform intact protein analysis using a LC-QTOF instrument.
The resulting molecular mass is 28479 Da (my protein is 28079 Da) and
consistent with all three sample preparation approaches.
Does anyone know why I got this result? I tried to google but failed to find
any useful information regarding the effect of vitamin C in LC-MS.
I really appreciate if someone can help on this.
Thanks! |
E***n
发帖数: 308 | 2 lost 4 amino acid under basic condition? check if there are any ASN at C or
N terminal?
just my guess, don't know munch about protein.
Good luck
approaches:
【在 b*****y 的大作中提到】
: My protein was eluted in 0.3% vitamin C in H2O and lyophilized (vitamin C
: was intended to prevent any Met oxidation of my protein). Since I know
: highly acidic pH is not good for LC-MS so I tried three different approaches:
: 1. directly dissolve in ammonium bicarbonate and adjust pH to 8
: 2. directly dissolve in ammonium bicarbonate and dialyzed against ammonium
: bicarbonate (3 changes in 6 hrs) and the final pH is 8
: 3. dissolve in ammonium bicarbonate and buffer exchanged to 3M guanidinium
: hydrochloride in Tris buffer and dialyzed against ammonium bicarbonate (3
: changed in 24 hrs). This approach should unfolds the protein at first and
: let it refolds.
|
h*****e
发帖数: 1330 | 3 Ascorbic acid 也就176，没到400，能不能整个内标看看 |
f*********e
发帖数: 1144 | 4 why u do this in basic condition??? don't understand the point of doing
buffer exchange to bring up pH. A simple control, don't do anything to the
protein, dilute in water or directly inject it, see what u got.
what column u are using? SEC or others?
400 difference could be a couple AA cleavages. you can check the terminus. or it lose some modifications.
approaches:
【在 b*****y 的大作中提到】
: My protein was eluted in 0.3% vitamin C in H2O and lyophilized (vitamin C
: was intended to prevent any Met oxidation of my protein). Since I know
: highly acidic pH is not good for LC-MS so I tried three different approaches:
: 1. directly dissolve in ammonium bicarbonate and adjust pH to 8
: 2. directly dissolve in ammonium bicarbonate and dialyzed against ammonium
: bicarbonate (3 changes in 6 hrs) and the final pH is 8
: 3. dissolve in ammonium bicarbonate and buffer exchanged to 3M guanidinium
: hydrochloride in Tris buffer and dialyzed against ammonium bicarbonate (3
: changed in 24 hrs). This approach should unfolds the protein at first and
: let it refolds.
|
m******u
发帖数: 340 | 5 The result is MW + 400. Not minus...
I think it is more likely adducts formed during ionization.
Well, I don't see the point to perform the test in basic condition either.
or it lose some modifications.
【在 f*********e 的大作中提到】
: why u do this in basic condition??? don't understand the point of doing
: buffer exchange to bring up pH. A simple control, don't do anything to the
: protein, dilute in water or directly inject it, see what u got.
: what column u are using? SEC or others?
: 400 difference could be a couple AA cleavages. you can check the terminus. or it lose some modifications.
:
: approaches:
|
b*****y
发帖数: 175 | 6 Thanks a lot for all your responses.
I did just inject the sample directly (highly acidic) and I did not have a
good spectra at all. The reason I am doing basic pH was based on our
previous experience and the recommended column conditions. Also I always
perform intact protein analysis and tryptic digestion peptide analysis
together. Trypsin works best at pH7-8 so I always try to use ammonium
bicarbonate to achieve this pH and have minimum salt after lyophilization.
I highly suspect vitamin C contributes to my weird results since I also
measured this protein when eluted without vitamin C and the molecular weight
was as expected (28079Da). So fat I see no instrument calibration problem.
I really appreciate if more input would be offered for this question. |
f*********e
发帖数: 1144 | 7 Haha, my mind was frozen...
Lz already sorts out it is present when use vc together. So it must be
something from vc. But it can't be resolved unless further detail info is
provided, such as protein sequence。what adducts could contribute to 400?
Complex glycosylation?
【在 m******u 的大作中提到】
: The result is MW + 400. Not minus...
: I think it is more likely adducts formed during ionization.
: Well, I don't see the point to perform the test in basic condition either.
:
: or it lose some modifications.
|
f*********e
发帖数: 1144 | 8 If u did pmf, what's the sequence coverage? Compare the one without vc and
the one with vc, if u really want to figure out the peak, do a differential
mapping. Use maldi for digest mapping would be much easier。
And what column are u using?
weight
【在 b*****y 的大作中提到】
: Thanks a lot for all your responses.
: I did just inject the sample directly (highly acidic) and I did not have a
: good spectra at all. The reason I am doing basic pH was based on our
: previous experience and the recommended column conditions. Also I always
: perform intact protein analysis and tryptic digestion peptide analysis
: together. Trypsin works best at pH7-8 so I always try to use ammonium
: bicarbonate to achieve this pH and have minimum salt after lyophilization.
: I highly suspect vitamin C contributes to my weird results since I also
: measured this protein when eluted without vitamin C and the molecular weight
: was as expected (28079Da). So fat I see no instrument calibration problem.
|
A****w
发帖数: 244 | 9 did you use EDTA during the protein prep?
Or just some modification to your sample.. |
b*****y
发帖数: 175 | 10 Thanks again for all your new inputs!
Additional information:
1. I used Vydac MS C18 capillary column.
2. The sequence coverage of my protein (from trypsin digested peptides MS
analysis) is consistent (80%) for both non-treated and treated samples. So
my confusion is since the protein identity is confirmed from peptide MS
analysis, why it does not show in protein MS analysis.
3. We do not have MALDI in the lab so it is hard to check using another MS
technique.
4. I found out the 28498 Da peak intensity in my vitamin C treated sample is
10x weaker than my non-treated sample, however, the protein peak (28080 Da)
is totally missing in my treated sample so the 28498 Da peak stands up in
the whole spectrum.
5. This protein is apolipoprotein A-I if someone knows more about it with
vitamin C. |
b*****y
发帖数: 175 | 11 As far as I know, no EDTA was used.
What would happen if EDTA was used?
【在 A****w 的大作中提到】
: did you use EDTA during the protein prep?
: Or just some modification to your sample..
|
