Preparation of Samples Prior To Mass Spectrometry

[Polyzoom]

Hey all,

I am trying to prepare samples for Mass Spectrometry. I know that desalting and protein concentration are important, but what else is important?

What buffers are compatible with mass spec?

thank U

[Polyzoom]

Also are there any straight and fast rules?
here are some that i know:

Avoid Buffers such as :
TRIS, CHAPS, PBS, and HEPES

Avoid Salts:
e.g. Na sodium, K potassium, P phosphate, citrate, perchlorate

Avoid surfactants and detergents

What is acceptable then?

[oBWhat]

Looks good so far. I always dialyze in Ammonium Bicarbonate. very compatible!

[MicroKiller]

For me.. i would jst freeze dry the sample then re-dissolve it in acetic acid.. if not then in 0.04% aqueous TFA.. If not you may try using a centrifugal membrane to desalt your sample and concentrate it down..

[aftabac]

Hi
i am giving below a link of virtual mass spectrometry laboratory. and here they have given the procedure for sample preparation in the form of video, i hope you will find it very informative for you.

[Only registered users see links. ]

best regards
aftab ahmad

[moleculardude]

Wow! great find Aftab!

[minhphd]

Hello every body.

I am a new member. My name is Minh. I come from proteomics Lab, Institute of Biotechnology, Hanoi, Viet nam.

I have some difficulties in a proteomics application to identify HBs Ag (from a recombinant vaccine sample). The sample comprises: HBs Ag 20ug/ml; Al(OH)3 < 1.25mg/ml; Themerosal< 0.1<%; formaldehyde<0.01%.
I tried several methods:
1. SDS-PAGE of 100ul sample (dryness), 1 observed band (Mw appro. 22kda) then was cut and followed by ingel-digestion and ESI-1DLCMS/MS identification (AB_QSTAR XL system).
2. Direct trypsin digestion (insolution digestion) of 100ul sample (dryness), following by ESI-1DLCMS/MS identification.
I spent more than 2 weeks on doing this work. now, I still do not identify the protein of interest.
I think that Al(OH)3 may impact on ability of trypsin digestion. But I don't know how to remove the impact.

Please suggest me how to solve this problem.
I am looking forward to hearing from you soon.

Thanks and best regards.

Pham Dinh Minh
Protein Biochemistry Lab, Institute of Biotechnology
Vietnamese Academy of Science & Technology

18 Hoang Quoc Viet Street, Cau Giay Dist., Ha Noi , Viet Nam

Tel: (+84)-4-7561903 Fax: (+84)-4-8363144 Emai: [Only registered users see links. ]

[molecule2005]

I would try dialyzing your sample Minphd in trypsin compatible buffer and trying digestion again and reanalysis.

Maybe your buffer is affecting the trypsin and/or the mass spectrometer machine.

[MicroKiller]

a suggestion to you.. You may consider trying to use the RapiGest solution from Waters.. it helps with the digestion..