Proteins & Mass Spectrometry
Mass spectrometry and tandem MS/MS
Alternate protocols from other
facilities, the ABRF discussion group, etc
-
ABRF In Gel
Digestion Protocol
- UCSF In Gel
Digestion Protocol
- ABRF
Discussion Thread about In-gel digestion
for Mass Spec
- Mass Spec directly
from electroblot membranes
- Passive
elution of whole proteins from SDS-PAGE
gels for subsequent Mass Spec analysis
- Staining and
excising spots
- Extraction
Method A - extraction into solvent for
~25 pmol or more protein per spot
- Extraction
Method B - extraction into MALDI matrix
for <<25 pmol protein per spot
- Alternate Extraction Method
In-gel Digestion protocols
Representative ABRF In Gel Digest
Protocol (copied from ABRF Archives
at
www.abrf.org)
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Kenneth Williams (Kenneth.Williams@yale.edu)
Thu, 06 Nov 1997 13:24:05 -0500
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Reagent"
In reponse to a query posted by Katheryn
Resing - this is the
representative in gel digest protocol
that will accompany samples that are
being distributed by the Internal
Protein Sequence Committee. We would
certainly welcome comments on the following
protocol.
Ken Williams
ABRF Internal Protein Sequence Research
Committee
Representative "In-Gel" Digestion
Protocol for Proteins in SDS PAGE Gel Slices
(10/97)
Samples to be digested in the gel are run in
as few lanes as possible to
maximize the concentration of the
protein within the bands of interest.
The gel is stained in 0.1% Coomassie
R250/20% MeOH / 0.5% AcOH, and then
destained in 30% MeOH until the bands
are visible and the background is
nearly clear. Volumes and reagent
quantities described herein are for one
band from a 1 mm gel slice digested
with trypsin. A reduction and
alkylation step is included.
Alternatively, the sample may be reduced and
alkylated prior to
electrophoresis. Note that an
alternate buffer system
is also provided for LysC digestion.
- Wash the gel slices for at least 1 hr
in 500 ml 100 mM NH4HCO3.
Discard the wash.
- Add 150 ml 100 mM NH4HCO3 and 10 ml 45
mM DTT. Incubate at 60oC for 30
min.
- Cool to room temp and add 10 ml of 100
mM iodoacetamide and incubate
for 30 min in the dark at room
temperature.
- Discard the solvent and wash the gel
slice in 500 ml of 50%
acetonitrile/100 mM NH4HCO3 with
shaking for 1 hr. Discard the wash.
Cut the gel into
2-3 pieces and transfer to a 200 ml
eppendorf style PCR tube.
- Add 50 ml of acetonitrile to shrink
the gel pieces. After 10-15 min
remove the solvent and dry the gel
slices in a rotatory evaporator.
- Re-swell the gel pieces with 10 ml of
25 mM NH4HCO3 containing Promega
modified trypsin (sequencing grade)
at a concentration such that a
substrate to enzyme ratio of 10:1
has been achieved. (If the amount of
protein is not known, add 0.1-0.2
mg of modified trypsin in 10 ml of 25 mM
NH4HCO3.) After 10-15 minutes add 10-20 ml
of additional buffer to cover
the gel pieces. Gel pieces need to
stay wet during the digest. Incubate 4
hrs to overnight at 37oC.
Proceed to step 8 if further extraction of
the gel is desired (recommended)
- otherwise continue with step 7.
- 0.5 ml of the supernatant may be
removed for MALDI analysis and/or the
supernatant acidified by adding 10%
TFA to a final concentration of 1% TFA
for injection onto a narrow- or
microbore reverse phase column. (If
necessary the sample's volume may
be reduced ~1/3 on a rotatory evaporator.)
- Extraction (Optional)- Save
supernatant from step 7 in tube X, and
extract peptides from gel twice
with 50 ml of 60% Acetonitrile/ 0.1% TFA
for 20 min. Combine all extracts in
tube X (using the same pipet tip to
minimize losses), and speed vac to
near dryness. Reconstitute in 20 ml of
appropriate solvent. Proceed with
chromatography or MALDI analysis.
Alternate Digestion Protocol.
If a LysC digest is desired a different
buffer system is used, using the
same volumes as before. This buffer
system (below) has been used
successfully with LysC from
Achromobacter lyticus. LysC from other
sources
has not been tested.
Wash the gel pieces in 0.5 M TrisHCl pH
9.2/50% acetonitrile.
Digest the protein(s) in the gel
slice with LysC in 0.1M TrisHCl pH 9.2.
UCSF In Gel digestion Protocol
(copied from UCSF page at
http://donatello.ucsf.edu/ingel.html
Return to Top of Page
This is the current procedure for In-Gel
digests used at UCSF (as of 10/11/99). It
has been refined to its
current state by contributions from
lab members over the last few years.
UCSF
In-Gel Digest Procedure
- Prepare the following solutions:
25 mM NH4HCO3 (100 mg/50 ml)
25 mM NH4HCO3 in 50% ACN
50% ACN/5% formic acid (may substitute TFA
or acetic acid)
12.5 ng/µL trypsin in 25mM NH4HCO3
(freshly diluted)
- Dice each gel slice into small pieces
(1 mm2) and place into 0.65 mL siliconized
tubes (PGC Scientific).
- Add ~100µL (or enough to cover) of
25mM NH4HCO3/50% ACN and vortex for 10
min.
- Extract the supernatant and transfer
to a separate tube (to be discarded).
- Repeat steps 3 and 4 two times. For
Coomassie blue stained gels, if gel pieces
are still very blue after 1st washing, you
can rehydrate the gel pieces with ammonium
bicarbonate before repeating the washes.
- Speed Vac the gel pieces to complete
dryness (~ 20 min).
- Estimate dried gel volume (V) in µL.
Add (3 x V) µL trypsin solution. This
volume will vary from sample to sample,
but on average ~25-60 µL is sufficient.
Vortex for 10 min.
- Incubate at 4 °C for 30 min. Add 25 mM
NH4HCO3 as needed to cover the gel pieces.
If trypsin solution remains in the sample,
pipet it off and replace with 25 mM
NH4HCO3.
- Spin briefly, cover tubes with
parafilm and incubate at 37C overnight
(16-20 hrs).
For low-level proteins (<1 pmol),
especially those separated by 1-D SDS-PAGE,
reduction and alkylation is recommended.
These procedures are performed after step 6.
- Prepare fresh solutions:
10 mM DTT in 25 mM NH4HCO3 (1.5 mg/mL)
5 mM iodoacetamide in 25 mM NH4 HCO3 (10
mg/mL)
- Add 25 µL (or enough to cover) 10 µM
DTT in 25 mM NH4HCO3 to dried gels. Vortex
and spin briefly. Allow reaction to
proceed at 56 °C for 1 hr.
- Remove supernatant, add 25 µl 55 mM
iodoacetamide to the gel pieces. Vortex
and spin briefly. Allow reaction to
proceed in the dark for 45 min. at room
temperature.
- Remove supernatant (discard). Wash
gels with ~100 µl NH4 HCO3, vortex 10 min,
spin.
- Remove supernatant (discard).
Dehydrate gels with ~100µL (or enough to
cover) of 25 mM NH4HCO3 in 50% ACN, vortex
5 min, spin. Repeat one time.
- Speed Vac the gel pieces to complete
dryness (~20 min). Proceed with trypsin
digest.
Extraction of Peptides
- Briefly vortex and spin the digest.
Add ~100 µL H2O, vortex 10 min, spin,
sonicate for 5 min. (Alternatively, add 5
µL H2O, vortex and sonicate. Remove 2 µL
and mix with 2 µL 30% ACN/5% formic acid.
Use 0.5 µL of the peptide mixture plus 0.5
mL DHB for preliminary analysis. Continue
extraction with remaining sample).
- Transfer the digest solution (aqueous
extraction) into a clean 0.65 mL
siliconized tube to which 5 µL 50%ACN/5%
formic acid has been added.
- To the gel pieces, add 50 µL (enough
to cover) of 50% ACN/5% formic acid,
vortex 10 min., spin, sonicate 5 min.
(only sonicate for the first organic
extraction). Pool extracted peptides
together in one tube. Repeat two times.
- Vortex the extracted digests, spin and
Speed Vac to reduce volume to 10 µL.
- Add 100 µL H2O, vortex, and Speed Vac
to ~10 µL. Add 2-5 µL 50% ACN/5% formic
acid.
- Utilize 1µL of the unseparated digests
for analysis by MALDI. Preliminary MALDI
results will determine whether or not a
cleanup using C18 ZipTips (Millipore) or
HPLC separation of the peptides is
required.
Matrices for unseparated digests:
a-cyano-4-hydroxycinammic acid in 50% ACN/1%
TFA (10 mg/mL).
2,5-dihydroxybenzoic acid (DHB), in 20%
ACN/1% TFA (10 mg/mL).
References:
Rosenfeld, et al., Anal. Biochem. (1992)
203, 173-179.
Hellman, et al., Anal. Biochem. (1995) 224,
451-455.
ABRF In-Gel Digests & Stains thread
an unedited discussion of various
issues of in-gel digestion
Return to Top of Page
JOHN@hoffman.mgen.pitt.edu (John Hempel)
This compiles related replies re. stains,
and details on in-gel Lys C digestions. -JH
:
From:
Ken_Mitchelhill@muwayf.unimelb.edu.au
10-NOV-1995 01:43:05.61
Ken Williams asked:
Has anyone carried out comparative testing
or know of any references to studies dealing
with the relative yields of peptides
obtained from in gel digests as a function
of protein stain. Has anyone
experience/references to results obtained
with in gel digests carried out on proteins
stained with Amido Black and/or other stains
besides Coomassie Blue R-250?
Ken, although you asked for details of
formal studies (of which I have seen none)
our anecdotal experience with hundreds of
in-gel digests is that the cleanest
chromatograms come from proteins stained
with Coomassie Blue G-250 and NOT Coomassie
Blue R-250. That was when we digested the
stained band, now that we destain the band
with acetonitrile/ammoniun
bicarb, I don't know that the same
observation applies but we still insist on
the G-250 stain. Regards to all.......Ken
Ken Mitchelhill John Holt Protein Structure
Laboratory St. Vincent's Institute for
Medical Research 41 Victoria Parade, Fitzroy
3065 Australia Voice (03) 9288 2497 Fax (03)
9416 2676 ken_mitchelhill.medicine@muwayf.unimelb.edu.au
From: Struktur@licr.uu.se" 10-NOV-1995
13:30:56.17
Dear Ken, Ken and others, I've
made 392 in gel digests over the last 3
years on samples from our Institute and many
others, where I can't control which Coomassie was used. However, we use R250
most of the time. Whenever there is a good
blue band, I get result; in other words I
don't feel that the yield is affected.
Background peaks may vary; therefore a
digest of a not completely destained gel
piece gives the non-peptide peaks. R250
elutes sharply during my wash of the SMART
C2/C18 2.1 mm column, via 10 minute gradient
from 40 to 80 % acetonitrile. Using multiple
wavelength detection, we usually disguise
junk-peaks (they often contain a high
proportion of aromatic absorbance.
Furthermore, as a measure of yield, I have
now and then re-digested (following the
protocol in my AB paper) the same gel piece
and never found extra material, so it feels
like dig and extraction are complete! Ulf
Hellman Ludwig Institute for Cancer Research
phone 46 18 550188 Fax 46 18 506867
From: Roland_S_Annan%notes@sb.com
10-NOV-1995 19:14:02.22
Ken Williams, Jim Vath at Genetics Institute has routinely
been doing in-gel digests followed by MALDI
from silver stained gels. Mathias Mann at
EMBL is also doing this routinely now. We
have tried it here and also found that it
works, but we don't use it routinely, yet.
Carlos Fernandez-Patron has published a
several papers on reverse staining
procedures using imidazole-zinc and in one
(Anal Biochem. 224, 203-211, 1995) has
performed in-gels digests on these bands. We
have not tried this. We will soon be trying
Stains-All and copper stain (also reverse
stain) in the not to far future. I will let
you know how it turns out. Roland Annan
Research Mass Spectrometry SmithKline
Beecham
From: PROTCHEM@serverdos.cigb.edu.cu
11-NOV-1995 19:47:49.74
To: Bryan Dunbar (P.
Fowler), Ken Williams and all. Hello! Here,
the Protein Chemistry lab at the Center for
Genetic Engineering and Biotechnology in
Havana. We have developed a high sensitive
procedure for the detection of proteins on
gels based on the principle of negative
staining (Lee, Dzandu, zinc and copper...).
The new procedure (Imidazole-SDS-Zinc
staining, Reverse Staining) is significantly
more sensitive than Coomassie blue, and is
specially conveived for further
microanalysis or activity, as it does not
modify the protein at all (as evidenced by
MS and other procedures). Reverse Staining
consists on the formation of an
imidazole-SDS-zinc 2 complex in the gel
matrix, deep white and insoluble, except in
the areas where proteins are, which remain
transparent, unstained. We currently use it
and it works well at the low picomole range.
As long as the gel is stained, proteins do
not difuse. (We sequenced a protein that was
kept in the RS gel for two years). In order
to get the protein amenable to further
analysis (on gel digestion, blotting,
elution...) they should be "mobilized" by
incubating the gel in a zinc chelating
solution. It can be applied to detect
Coomassie blue undetected proteins (i.e.
those below the detection limit of CB or
those with low affinity for CB). Detailed
protocols are published in:
- BioTechniques vol 12 no.4, 564-573 (1992)
(the principles of the method and
applications)
- Febs vol 296, 300-304 1992 (extension to
gels without SDS, increase in sensitivity,
applications)
- Anal. Biochem. 224, 203-211 (1995) (strategy
for protein purification based on single
band transfer from RS gels, applications)
- Anal. Biochem. 224 263-269 (1995) (double
staining of CB-stained gels by
imidazole-SDS-zinc RS: sensitive detection
of CB undetected proteins.)
- Electrophoresis vol 16, 911-929 (1995)
(single step electrotransfer of reversed
stained proteins on gels onto reversed phase
matrix) (a direct link between gel
separation and HPLC by blotting onto a
reversed phase cartridge).
We will highly appreciate the comments of
all users in order to further develop these
procedures. Lila Castellanos-Serra Head
Prot. Chem. Dept. Division of Physical
Chemistry CIGB Havana Fax: 053 7 33 60 08
053 7 21 80 70 email protchem@serverdos.cigb.edu.cu
padron@serverdos.cigb.edu.cu
From: Struktur@licr.uu.se 16-NOV-1995
18:40:14.
We store Coomassie stained gelpieces in
Eppendorf tubes, just as the gel pieces come
from the destained gels, i. e. without any
liquid. We are even ignorant enough to store
them at RT, sometimes for months, and have
had no problems so far. Should they happen
to get dry - no worries! Storing in liquid
have at least the potential risk that the
protein might "swim out". And why would you
make a tryptic digest when Lys C (WAKO
stuff) works so excellent?? Fewer and longer
fragments! We have almost totally abandoned
trypsin; we use it only for comparative
peptide maps. Lisa Bibbs and Gary Hathaway
asked for my protocol on using LysC for in
gel digestion. Here it goes:
- Wash the gelpiece(s) twice with 0.5 M
TrisCl pH 9.2/50 % acetonitrile at 30
centigrades for ca 45 min. Discard washings.
- Dry completely using a gentle stream of
nitrogen or SpeedVac.
- Add to the dry gel 0.5 microgram of LysC
in 10 microliter of 0.1 M TrisCl pH 9.2.
- Continue adding 0.1 M TrisCl pH 9.2 in
aliquotes until gel has fully reswollen.
- Incubate at 30 centigrades overnight.
- Save liquid in fresh Eppy.
- Add to the gel ca 150 microliter (dep on
gel volume) 0.1 % TFA/60 % acetonitrile.
Incubate at least 60 min. Repeat and combine
extracts into the Eppy in #6 above.
- Reduce volume (and acetonitrile!) by
SpeedyVac. We remove ca 2/3.
- Isolate fragments by narrow-bore RPC. We
use the "=B5RPC C2/C18 SC 2.1/10= " in
Pharmacia'a "SMART", utilizing a very
shallow gradient (0.25 %/min).
LysC alone elutes somewhere after 40 %
acetonitrile. We seldom see any
autodigestive products; at least we have
NEVER sequenced any! I hope you will be
happy with this protocol; we have used it
for about 9 months now. Compared to trypsin,
the yield is at least as good, if not
better. We have never seen any "wrong" cuts;
therefore we dare to "add" a Lys BEFORE
every peptide sequence we determine. And
usually we see the last Lys in peptides.
Ulf Hellman Group Head Assoc. Member Ludwig
Institute for Cancer Research Box 595 S-751
24 Uppsala Sweden Phone 46 18 550188 =46ax
46 18 506867 E-mail
Ulf.Hellman@LICR.uu.se
MassSpec:
MALDI from blots
Return to Top of Page
Does somebody know a method or have some
suggestions, how to do
MALDI on proteins blotted to PVDF membranes
and CBB-stained.
Arne,
Dr Christoph Eckerskorn is one of the best
authorities on this subject. His
email is eckerskorn@genmic.biochem.mpg.de (Christoph
Eckerskorn).
From his talks, I think you will find it is
bad news to stain the blot at
all. It is best to keep it wet and locate
the protein by staining a side
strip. A literature search for Eckerskorn
and F. Lottspeich will produce
appropriate papers, but here's a recent one
TI: ULTRAVIOLET MATRIX-ASSISTED LASER-DESORPTION
IONIZATION-MASS SPECTROMETRY OF ELECTROBLOTTED PROTEINS
AU: SCHREINER_M, STRUPAT_K, LOTTSPEICH_F,
ECKERSKORN_C
JN: ELECTROPHORESIS, 1996, Vol.17, No.5,
pp.954-961
AB: Direct mass spectrometric analysis of
proteins electroblotted onto
polyvinylidene fluoride membranes after
sodium dodecyl sulfate-polyacrylamide gel electrophoresis is
demonstrated by matrix-assisted
laser desorption ionization-mass
spectrometry (MALDI-MS) with a
linear time-of-flight instrument, equipped
with a nitrogen laser (337nm). The blotted proteins were desorbed
directly from the blotting
membrane after incubation with sinapinic
acid as matrix. Different
commercially available membranes resulted in
high quality protein
signals for hydrophobic membranes exhibiting
high specific surface
areas (Immobilon PSQ or Trans-Blot) of for
charged membranes (Immobilon CD). Systematic investigations
with standard proteins were
performed to compare standard preparation
procedures for ultraviolet
(UV) MALDI-MS on stainless steel sample
stages and preparation of
proteins immobilized onto membranes either
by direct application from
protein solutions (spotting) or by
electrotransfer from gels
(electroblotting). Aspects such as mass
resolution, reproducibility
from shot to shot and spot to spot, mass
accuracy, and preservation
of protein localization are addressed in
this paper
Good luck
Len
******************************************************
Dr Len C. Packman
Assistant Director of Research
Protein and Nucleic Acid Chemistry Facility
Department of Biochemistry, Tennis Court
Road, Cambridge, CB2 1QW, UK
Tel: +44 (1223) 333639 (including
answerphone)
FAX: +44 (1223) 333345
e-mail: lcp2@mole.bio.cam.ac.uk
Visit my WWW page at http://www.bio.cam.ac.uk/proj/adr/PNAC/pnac.html
Elution of proteins
from SDS-PAGE gels for subsequent Mass Spec
Return to Top
of Page
from Cohen, S. L.
and B. T. Chait (1997). “Mass spectrometry
of whole proteins eluted from sodium dodecyl
sulfate- polyacrylamide gel electrophoresis
gels.” Anal Biochem 247(2): 257-67.
Gel Staining and Excision
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Immediately following electrophoresis, the
gels can be rinsed in water, although we
recommend against rinsing when handling
low-molecular-mass proteins (<20 kDa) that
are at low picomole gel-loading amounts.
Protein bands were visualized by soaking the
entire gel in 45 µl copper-staining solution
(supplied 10x from Bio-Rad) for 5 min with
gentle rocking (22). Alternatively,
zinc-staining (23) or imidazole-zincstaining
(24) protocols can be used. After staining,
the gels were thoroughly rinsed in deionized
water. Because copper and zinc staining are
negative stains, protein bands appear
translucent and are best viewed against a
black background. Bands with as little as 17
ng (~ 1 pmol) of myoglobin or recombinant
leptin were visualized by copper staining.
Gel Excising and
Destaining (Fig. 1)
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browser)
Destaining was performed on
individual gel slices containing a single
protein band as depicted in Fig. 1 above.
Figure 1, step 1. Protein bands were excised
from the stained gel with a clean, sharp
straight-edge razor (Fig. 1).
The gel slices were excised as close as
possible to the boundaries of the protein
band. The resulting blue-tinted gel slices
typically had the dimensions 1 to 1.5 mm x
6-7 mm and were best handled with flatend
tweezers. Protein extraction should be
performed immediately after destaining. If a
delay is anticipated, it is best to leave
the gel slices stained and stored moist (but
not soaking) at 4°C in a sealed microtube
until needed.
Step 2. The destaining process completely
removes the visualization stain as well as
the SDS detergent (22). Each gel slice was
treated in a separate 1.5-ml microcentrifuge
tube using standard copper-destaining
protocols (Bio-Rad). Zinc-stained gels can
also be destained using the same protocols.
Destaining was performed by a three-step
soaking process using a copper Tris-glycine
destain solution (Bio-Rad). The first
soaking of the gel is carried out in a
solution consisting of 100 µl destain buffer
and 900 µl water. The tube is sealed and
vortexed (Tomy MT-360 36-sample microtube
mixer; Tomy Tech, Palo Alto, CA) for 5 min
at room temperature. During the first
destaining period the solution turns pale
translucent blue (for copperstained gels)
and may become slightly foamy. The gel will
retain a faint blue tint. The second soaking
is performed in fresh solution of 100 µl
destain and 900 µl water with 10 min of
vortexing. At the end of the second soaking,
the destain solution and gel will be nearly
colorless. The final soaking is performed in
a solution of 50 µl destain and 950 µl water
with 5 min of vortexing. At the end of the
third soaking, the destain solution as well
as the gel are clear. The destained gel
pieces are rinsed in deionized water and are
ready for extraction.
Step 3. Prior to protein extraction, the
gels are crushed. Before crushing, the
destained gel slices are gently blotted free
of any excess water clinging to the gel with
a lint-free tissue, a step that facilitates
crushing of the gel. With the aid of
tweezers, the gel slices are placed into a
0.65-ml polypropylene microcentrifuge tube (PGC
Scientifics, Gaithersburg, MD). The
microtube should be of high quality, free of
plasticizers and contaminants that can
compromise high sensitivity MS measurements.
From our experience, we avoid the use of
colored microtubes. The gel slice is
manually crushed at the bottom of the
microtube for a few seconds with a
sharp-pointed dental tool. Mechanical
homogenization of the gel can be used
instead of the manual crushing, but we
recommend against it since the resulting
small gel pieces tend to clog pipettor tips.
At this point, either of two methods
described below (Extraction Methods A or B)
can be used to extract the protein.
Extraction Method A (Fig. 1)
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This method of extraction elutes protein
directly into an aqueous solution and is
best suited to gel loadings exceeding 25
pmol. Solution compositions that we have
examined for protein extraction include
formic acid/ water/2-propanol (1:3:2 v/v/v)
(FWI), formic acid/water (1:5 v/v),
water/2-propanol (2:1 v/v), and pure water.
Optimal extraction efficiency occurred with
the FWI combination, whereas no extraction
was observed with pure water. (The relative
extraction efficiencies are based on a
qualitative assessment of the MALDI-MS
signal-to-noise ratios). Other acids and
water-miscible organic solvents (e.g., 0.1%
TFA and acetonitrile) can be used for
extraction.
Figure 1, step 4a.
Sufficient extraction solution (~30-40 µl)
is added to the crushed gel to completely
cover the gel pieces.
Step 5a. The tube is closed and vigorously
shaken or vortexed at room temperature from
4 to 8 h. Although not essential for protein
elution, shaking/vortexing facilitates
protein extraction. Generally, the greater
the protein loading or the smaller the
protein, the shorter the vortexing time
required for extraction.
Step 6a. After the vortexing period, the
microtube is centrifuged and the supernatant
is retrieved with a 10-µl micropipettor,
avoiding taking up any of the gel pieces.
The crushed gel is washed once with an equal
volume of fresh extraction solution and the
wash is combined with the supernatant.
Steps 7a and 8a. If the protein is to be
analyzed by MALDI-MS, the extraction
solution is lyophilized and a MALDI matrix
solution (2 µl for Fig. 2) is added to
redissolve and retrieve the protein for MS
analysis using the dried-drop method of
matrix crystallization (see below). The
matrix solution is FWI saturated with
4-hydroxy-a-cyano-cinnamic acid (4HCCA)
(25). If the protein is to be analyzed by
ESI-MS, the extraction solution is injected
into a protein-desalting cartridge (Michrom
BioResources, Inc., Auburn, CA) that is
mounted to the ESI capillary inlet. The
loaded cartridge is flushed several times
with a 2% acetic acid wash solution.
Following the washing, the protein is eluted
from the cartridge into the electrospray
source with a solution consisting of 70%
acetonitrile/2.5% acetic acid at 6 µl/min.
ESI-MS was performed on a TSQ-700 triple
quadrupole (Finnigan Corp., San Jose, CA)
using standard ESI conditions for analyzing
proteins (26).
Extraction Method B (Fig.
1)
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This method of extraction elutes protein
from gels directly into a MALDI-MS matrix
solution and is best suited to gel loadings
below 25 pmol of protein or for proteins
that fail to passively elute using
Extraction Method A (described above). In
Method B the matrix is allowed to undergo
slow crystallization (18) during the protein
extraction period. The matrix used
throughout was 4HCCA, although sinapinic
acid can also be used. The moderately low
solubility of these cinnamic acid
derivatives in the matrix solution is an
important characteristic for efficient slow
crystallization (18). The most frequently
used matrix solution in our experiments
consisted of a saturated solution of 4HCCA
in FWI. Other solvent systems were also
examined (e.g., 0.1% TFA:acetonitrile, 2:1
v/v) (25). Gel extraction is as follows:
Figure 1, step 4b. After
crushing the destained gel (see above),
enough matrix solution (~30-40 µl) is added
to cover the gel pieces.
Step 5b. The tube is closed and vigorously
shaken or vortexed at room temperature for
1-2 h.
Step 6b. Matrix crystallization is induced
by leaving open the top of the microtube
(0.5-1 h) with vortexing. The matrix
solution will become slightly cloudy with a
suspension of 4HCCA crystals. An aliquot (1
µl) of the milky supernatant can be
immediately retrieved and deposited onto the
MALDI probe for MS analysis while the
remaining sample is closed and left
vortexing for additional extraction. Avoid
taking up any gel pieces while pipetting and
avoid crushing the matrix crystals once they
are on the probe.
Steps 7b and 8b. If the MALDI-MS response
from the "immediately retrieved" crystals is
weak, the signal should improve from
crystals that have undergone a longer period
(~4-24 h) of vortexing in the gel/matrix
suspension. During extended vortexing
periods, the matrix crystals precipitate to
the bottom and/or cling to the sides of the
microtube and can be retrieved with a
micropipettor for MALDI-MS analysis. Also,
during the vortexing period volume losses of
the solution due to evaporation and aliquot
sampling may occur. To offset the volume
losses, small amounts (5-10 µ) of
matrix-free extraction solution are added to
keep the gel pieces completely submerged in
solution. For the results given in this
paper vortexing times ranged from 6 h (Fig.
5, see original paper) to 12-19 h (Fig. 6,
see original paper).
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L. Castellanos-Serra
(protchem@cigb.edu.cu)
Mon, 18 May 1998 09:28:57 EST
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Dear Amanda
Working at the low picomol level we get high
yields by this passive elution procedure:
Stain the gel by negative staining (Cu, Zn
or for higher sensitivity, ImH-Zn). Chelate
the metal (this is crucial!) by 2 x 5 min.
incubation in Tris, 0.2-0.4M Gly, pH 8.0
(about 1 ml per band). Wash a few seconds
with 1 ml water. Crush the gel band through
a metal sieve placed in a 1 ml syringe, to
get about 32 micrometer particles. Incubate
with moderate vortexing, 2 x 10 min in
Tris-Gly or ammonium bicarbonate (30-50 mM,
pH 8.0, do not add detergent at all) and 1 x
0.5 min in water. Collect each time by
centrifugation ( a few seconds). Each time
the elution volume should be about twice the
volume of the crushed gel. Note that this
procedure does not work with CB stained
bands, only with negative stain. Proteins
reduced in bME before electrophoresis tend
to reaggregate on gel causing low elution
yields; thus, load in a sample buffer
without bME or fully reduce and alkylate the
sample in 8M urea before electrophoresis.
(Ref. J. Prot. Chem., 16, 415-419, 1997,
Electrophoresis, 17, 1564-1572, 1996)
Lila Castellanos-Serra
<protchem@.cigb.edu.cu>
Prot. Chem. Dept.
Div. Phys. Chem. CIGB
FAX: 537-218070
Havana Cuba
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