I'm facing a problem with respect to the protein loss after acetone precipitation, followed by its insolubility in the 100mM Ammonium bicarbonate solution, I also tried dissolving the protein pellet by increasing the pH by usage of 8M urea but I feel that urea is not a good option when it comes about loading the protein sample in nano lc ms ms. I carried out the protein digestion very carefully and then determined the protein concentration again and found out that there was a huge protein loss there. Now i'm unable to retaliate the problem and the low protein yield is ultimately affecting proteomic result. Could you please suggest me some other alternative? It would be really appreciable
Related to the protein precipitation and its re-suspension in a suitable buffer?
First of all I want to ask you about your protein:
What kind of protein are you studying? Have you purified it?
Secondly, for the protein precipitation may be you could precipitate it by adding 4 volume of ethanol over/night at -20°c centrifuge and dissolve the pellet and dissolve it ammonium acetate buffer and dried your pellet, In that case the buffer will be evaporated, to ovoid ammonium which could interfere in protein dosage as Urea (Bradford method)
However we have done the same work to staudy a glucanase protein:
Our purified protein was sparated by SDS PAGE and the band was extracted from the gel and treated by nano lc ms ms technique.
So you can dissolve your protein pellet in in SDS-Buffer to carry out SDS PAGE (This means that you can identified your protein in the gel) to extract it from the gel.
I send you our work published in Moleccular biotechnology 2014
Biochemical Characterization, Molecular Cloning, and StructuralModeling of an Interesting b-1,4-Glucanase from SclerotiniaSclerotiorum
Haifa Chahed, Aymen Ezzine, Amine Ben Mlouka, Julie Hardouin, Thierry Jouenne and M. Najib Marzouki (2014)
MolecularBiotechnology Volume 56, Issue 4, pp 340-350.
I also send you our protocol:
Mass Spectrometry Analysis
Label-Free Technique
About 30 micro-g of proteins (from the crude extract) were
resolved by 1D SDS-PAGE 7 % and then briefly stained
with Coomassie Blue. After destaining the gel, slices were
excised from each lane and subjected to tryptic digestion.
In brief, the disulfide bonds were reduced by the addition of
dithiothreitol and the free cysteine residues were alkylated
with iodoacetamide. The gel plugs were washed prior to a
dehydration step, followed by the tryptic digestion. The
resultant tryptic peptides were finally lyophilized and ready
to use for the spectrometry analysis.
NanoLC–ESI-MS(n) Experiments
All experiments were performed on a LTQ-Orbitrap Elite
coupled with an Easy nLC II system (both from Thermo
Scientific). For mass spectrometry analysis, peptides were
dissolved in 0.1 % formic acid. After sample loading onto
an enrichment column (C18 PepMap100, Thermo Scientific),
the separation was achieved with an analytical
column needle (NTCC-360/100-5-153, NikkyoTechnos,
Japan). The mobile phase consisted of H2O/FA 0.1 %
(buffer A) and ACN/FA 0.1 % (buffer B). Tryptic peptides
were eluted at a flow rate of 300 nl/min using a three-step
linear gradient: from 2 to 40 % B over 105 min, from 40 to
100 % B in 4 and 15 min at 100 % B. The mass spectrometer
was operated in positive ionization mode with a
capillary voltage and a source temperature set at 1.5 kV
and 275 _C, respectively. The mass spectrometer was
operated in the data-dependent mode to automatically
switch between MS (from m/z 400 to 2,000 with a
resolution of 60,000) and MS/MS acquisitions.
Sir,
The protein isolated from MIN6 cell lysate (lysis by RIPA Buffer)
And the protein is not a purified protein.
Gave a try with amonium sulphate precipitation under chilled condition.after that dialyze the sample in the same buffer in which crude protein is dissolved.
Reconstituting precipitated proteins is often difficult. Time and very gentle agitation (e.g., an end over end rocker) often work, but sometimes surfactant is needed. If you don't want to deal with surfactants, which can be problematic for downstream processing, then you might try non-detergent sulfo-betanes (NDSBs). These can be used in place of surfactants to help solublize proteins, yet don't bind directly to the protein so can be readily removed. Since they are amphilphilic they don't effect electrophoresis or pH significantly. But you do have to use them at high concentrations (e.g., 0.5 M) and they aren't cheap.