While detergent removal can be accomplished by HILIC (or the Michrom NID
column as published in the ABRF article as referred to by Alpert) the
problem HILIC presents is the high organic concentrations required to bin=
d
some proteins. If your protein requires detergent for solubility, maybe
high MeCN isn't so bad an approach, but the limit of solubility needs to =
be
considered. Hence, this is not as universal a system as implied above.
Additionally, the use of the Amika UltraMicrospin columns with MeCN is NO=
T
recommended at this time due to MeCN extractables from their housings.
(When they change their materials of construction, it would be appropria=
te
to use that product. There isn't a problem with water or MeOH though.).
In the interim, for silica based packings, either use their MacroSpin
columns loaded with smaller amounts of packing to make them operate like
the UltraMicrospin.
Better yet, they have a Detergent UltraMicrospin column which comes in a
variety of formats (Spin, Tip or 96 Well plate) which does not require
organic solvents to remove the nonionic detergent. To use Amika's
Detergent cartridge, put buffer (tris or digestion buffer 10-15 mM) on th=
e
column to condition it, then spin it out. Next apply the sample and
incubate the sample at 37C for 15 min.. Then spin the protein away from t=
he
detergent (If using their Tip format use the pipetter to move the liquid.=
).
Another, although more specific, alternative for detergent removal from
proteins would be to use an ion exchange cartridge as appropriate for the
pI to bind the protein and let the nonionic detergent pass through the
cartridge with buffer, either at the loading step or with an intermediate
wash step. Elution in 0.5 M salt would then allow subsequent RPC analysi=
s
directly into the MS (Use your switching valve to dump the salt at the
solvent front into waste!).
Call me if you need more specifics on any of the above.
Sincerely,
Amos Heckendorf
The Nest Group, Inc.
508-481-6223
-----------
> Dear ABRF-members,
>
> we have encountered problems with the MS-analysis of some samples from
> in-gel digestions of 2DE spots. These samples (1-5 pmoles) contain
> components eluting from the HPLC separation as a ladder in the middle =
of
> the gradient (Pepmap. C180.3x250 mm, 30-45% ACN). The components have =
an
> increasing mass difference of 44 Da relative each other. We have
> identified them to originate from non-ionic detergents containing PEG
> moieties (e.g. Triton X-100).
>
> I searched the ABRF archive and found that byproducts (aldehyde and
> peroxide) in PEG products also might cause problems with the digestion
> itself.
> We have tried to remove these agents using an NID-trap (Michrom
> Bioresources). In our hands, however, this separation doesn't seem to
> work too well for digest mixures. The peptides seems to go with the vo=
id.
>
> If you need to use non-ionic detergents, does anyone have any good
> protocols for removing the problems we see, e.g. removing non-ionic
> detergents and/or byproducts therof, from digestion mixures?
>
> ----------------------------------
> Erland Holmberg, PhD
> Manager, Protein Identification
> Research, Pharmacia & Upjohn, Sweden Tel: +46 8 695 9094
> Mobile +46 8 817 0619
> Fax: +46 8 695 7640
>
> e-mail:erland.holmberg@eu.pnu.com
> ----------------------------------
>A good general protocol for getting nonionic detergents out of samples i=
s to
>use Hydrophilic Interaction Chromatography (HILIC). This was discussed =
in the
>Dec. '97 issue of ABRF News; see also Jeno et al., Anal. Biochem. 215 (1=
993)
>292. To get the detergents out of a digest on a small scale, add 5 volu=
mes of
>acetonitrile to the sample and apply it to a small column of a very pola=
r
>material. An example is the ultramicro spin cartridges of Amika packed =
with
>PolyHYDROXYETHYL A; these can run as little as 5 ul of sample. The cart=
ridges
>fit into a microcentrifuge tube and sequential washes are performed in a
>microcentrifuge. Detergents aren't retained and pass right through the
>cartridge, while virtually all peptides are retained. You can then elut=
e the
>peptides with a step to water. The cartridges can be reequilibrated wit=
h 85
>or 90% acetonitrile and reused.
>
>Andy Alpert
>PolyLC Inc.
------------------
Amos Heckendorf (nestgrp@world.std.com)
The Nest Group, Inc., Value Added Resellers of HPLC Columns (Vydac=81,
PolyLC=81, Hydrocell=81, BioChrom Labs=81, Jones, Jordi-Gel=81, Nucleosil=
=81, Higgins
Analytical=81) and Isolute=81 SPE cartridges; ESA=81 Electrophoresis PAGE
mini-gels and Sialomed=81 COZAP Destaining pads and MicroDialysis tools; =
and
Quantum Magnetics=81 Immunomagnetic Affinity Particles . Tel: 800-347-63=
78
or 508-481-6223; FAX 508-485-5736;
45 Valley Rd, Southboro, MA 01772-1306
HPLC Applications and Protocols at:
HTTP://world.std.com/~nestgrp/protocols/protocol.html