Protocols I

1
Protocols I Gel stainingMany staining methods
are compatible with subsequent tryptic digestion
and mass spectrometric analysis. However, it is
important that the proteins are not covalently
modified or irreversibly fixed in the gel during
staining. We prefer that you stain the gels by
Coomassie blue stain (Bio-Safe) from Bio-Rad,
Cat 161-0786. If you choose to use silver
staining, be careful to follow the suggested
protocol below. Otherwise, it may not be possible
to obtain any mass spectrometry data. Coomassie
Blue staining Wash 3 5 min washes with ddH2O
Fix the gel with 40 methanol, 10 acetic acid,
and 50 H2O for 60 min Rinse the gel with ddH2O
for 3 times Wash with 50 ethanol, 50 H2O
overnight to decrease the gel background at 4oC
Rinse with ddH2O 3 times Stain the gel in
Bio-SafeTM coomassie stain solution which is
enough to cover the gel Gently shake for 1 hour
Rinse with ddH2O for 3 times Wash with ddH2O
overnight Wrap the gel with clean Sara wrap.
Acquiring gel image can be done with a scanner.
Never touch your gel with bare skin. Store the
gel in 1 to 2 acetic acid Please contact us if
you prefer to use other stain like SYPRO that is
compatible with mass spectrometry. Silver
staining (Adapted from Blum et al,
Electrophoresis, 8, pp93-99, 1987 and
Schevchenko, A. et al. (1996) Anal. Chem. 68,
850-858) Fix the gel in 50 ethanol, 5 acetic
acid for 1hr. Wash the gel in 50 ethanol
overnight. Wash gel in Milli Q H2O for 310 min
Sensitize gel in 0.02 Na2S2O3.5H2O for 1 min.
Wash gel in chilled H2O, 330 secs Incubate gel
in cold 0.1 AgNO3 for 20 mins. at 4ºC Wash the
gel in H2O 320 secs Transfer the gel to a clean
container Wash the gel in H2O for 1 min.
Develop the gel in 0.05 Formalin, 3 Na2CO3 (To
make 500ml of 0.05 formalin needs 0.25 ml of 37
formaldehyde) Change the developing solution
when the developer turns yellow. Stop the
staining with 5 acetic acid Wash the gel 3 10
mins with ddH2O Store the gel at 4º C in 1 HAc
TCA PrecipitationDilute protein to 500 ul with
water in eppendorf tube Add 50ul of DOC 25
ul of TX 100 80 ul of TCA (13TCA) At least 2
hrs on ice Spin in a bench-top microfuge at 4 oC
for 30 min at Max. speed (15,000 rpm). There
will be a flaky pellet up the side of the tube
Dump sup, let the tube stand on end on a kimwipe
to get most of the liquid out. Don't sweat it if
a little is left in the bottom Add 500ul of
ethanol/ether( cold aceton) . Bath sonicate to
thoroughly disrupt pellet On ice for 30 min
Repeat step 4 There will be little to no pellet
Repeat step 6 Let air dry at room temperature
Re-suspend samples in 5 ul running buffer first,
then 20 ul sample buffer Heat at 85 degree for 5
min. Load sample to each well (25ul)
2
References Concentration and detergent
removalSauve, D. M., D. T. Ho, et al. (1995).
"Concentration of dilute protein for gel
electrophoresis." Anal Biochem 226(2)
382-3.Wessel, D. and U. I. Flugge (1984). "A
method for the quantitative recovery of protein
in dilute solution in the presence of detergents
and lipids." Anal Biochem 138(1) 141-3. Protein
Precipitation by Trichloroacetic AcidBrown, R.
E., K. L. Jarvis, et al. (1989). "Protein
measurement using bicinchoninic acid elimination
of interfering substances." Anal Biochem 180(1)
136-9. In-gel Trypsin Digestion Procedure To
avoid keratin contamination, wear gloves
throughout this process. 1. Place the gel on a
stringently cleaned glass plate and excise the
gel band of interest out of the gel using a clean
razor blade. Cut the band into the
smallest pieces possible. 2. Place the gel pieces
into a centrifuge tube and cover with
acetonitrile for approximately 15 minutes at room
temperature. Spin the tubes, remove
the acetonitrile by pipette, and dry the gel
pieces by Speed-Vac. 3. Re-swell the dried gel
pieces in buffer containing trypsin in 50mM
NH4-HCO3 at 4C for 45 minutes. Add enough buffer
so the gel pieces are just covered. The suggested
amount of trypsin is 12.5 ng/ul of buffer for
proteins that have been silver stained. 4.
Incubate the swelled gel pieces in trypsin buffer
at 37C for at least 3 hours (ideally
overnight). 5. Centrifuge the gel pieces and
collect the supernatant. 6. Add 25mM NH4-HCO3 to
the gel pieces and soak for approximately 25
minutes. 7. Further extract the peptides by
soaking the gel pieces in 5 formic acid in
50 acetonitrile 45 water for 30 minutes,
followed by spinning and collection of
the supernatant. Repeat 3 times. 8. Dry the
collected supernatants by Speed-Vac until the
desired volume has been reached. In-Solution
Trypsin Digestion Method 1 The sample should
contain a minimal amount of DTT or detergents for
the following method to work. 1. By Speed-Vac,
dry the protein sample in a microfuge tube. 2.
Dissolve the proteins in a minimum of 20ul of
freshly made 0.1M ammonium bicarbonate. Try to
keep the volume of the digest solution as small
as possible. 3. Denature proteins by incubating
at 55C for 30 minutes. 4. Add trypsin to the
sample to a trypsin sample ratio of 1100. 5.
Incubate at 37C overnight. 6. Digestion can be
confirmed as a disappearance of the corresponding
protein band on protein SDS-PAGE. In-Solution
Trypsin Digestion Method 2 This procedure is
routinely used in the lab after ICAT labeling
because it allows for some DTT or detergents,
though method 1 results in better digestion. 1.
Dilute the sample to a maximal detergent
concentration of 0.01 SDS. 2. Add trypsin to a
trypsin sample ratio of 120. 3. Incubate at
37C overnight. 4. Digestion can be confirmed as
a disappearance of the corresponding protein
band on protein SDS-PAGE.
3
Protocols II n-Gel Digest Procedure 1.Wearing
gloves and sleeve protectors, wipe down ALL
surfaces in the hood with methanol/water
moistened lint-free cloth, including the
outside of all your tubes (make sure to not wipe
off the labeling!), the outside and inside of
the Speed Vac and centrifuge, tube racks, bottles
etc. Wipe razor blades with methanol-soaked
lint-free cloth. 2. 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)
3. Dice each gel slice into small pieces (1 mm2)
and place into 0.65 mL siliconized tubes (PGC
Scientific). 4. Add 100µL (or enough to cover)
of 25mM NH4HCO3/50 ACN and vortex for 10 min.
5. Using gel loading pipet tip, extract the
supernatant and discard. 6. Repeat steps 3 and 4
once or twice. 7. For low-level proteins (lt1
pmol), especially those separated by 1-D
SDS-PAGE, reduction and alkylation is
recommended. These procedures are performed after
step 6. Prepare fresh solutions10 mM DTT in 25
mM NH4HCO3 (1.5 mg/mL)55 mM iodoacetamide in 25
mM NH4 HCO3 (10 mg/mL) Add 25 µL (or enough to
cover) 10 mM 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. 8. Speed Vac the gel pieces to complete
dryness ( 20 min). 9. Proceed with trypsin
digest Add trypsin solution to just barely
cover the gel pieces. Estimate the gel
volume and add about 3x volume of trypsin
solution. This volume will vary from sample
to sample, but on average 5-25 µL is sufficient.
Rehydrate the gel pieces on ice or at
4C for 10 min. Spin. Add 25mM NH4HCO3
as needed to cover the gel pieces. Spin
briefly and incubate at 37C for 4 hours -
overnight. Extraction of Peptides 1.Transfer
the digest solution (aqueous extraction) into a
clean 0.65 mL siliconized tube. 2.To the gel
pieces, add 30 µL (enough to cover) of 50 ACN/5
formic acid, vortex 20-30min., spin, sonicate
5 min. 3. Repeat. 4. Vortex the extracted
digests, spin and Speed Vac to reduce volume to
10 µL. 5. Either proceed with C18 ZipTip
(Millipore) cleanup or analyze with LC-MS.
Add 2-5 µL of 5 formic acid. When analyzing
low levels of protein, concentrate the petides by
eluting from ZipTips using 3µL of elution
solution, into a clean 0.65 mL siliconized tube.
Utilize 1µL of the unseparated digests for
analysis by MALDI. Matrices for unseparated
digests a-cyano-4-hydroxycinammic acid in 50
ACN/1 TFA (50 mg/mL). 2,5-dihydroxybenzoic acid
(DHB), in 20 ACN/1 TFA (50 mg/mL). References
Rosenfeld, et al., Anal. Biochem. (1992) 203,
173-179.Hellman, et al., Anal. Biochem. (1995)
224, 451-455.
4
Protocols III Protocol for Gel Fixing, Staining,
and Destaining (Coomasie) 1. To fix gel soak in
25 isopropanol/10 acetic acid/65 Milli-Q
water for 20 minutes for 1-mm thick gels 2. To
stain gel Use BioRad R-250 Coomassie 0.01 IN
10 acetic acid, and stain overnight on an
orbital shaker at 40 rpm. 3. To destain Pour off
Coomassie solution from step 2 and replace
with 10 acetic acid, and continue gentle shaking
at 40 rpm. 4. As solution turns blue, replace
with fresh until background is destained (protein
bands should remain stained). 5. Leave gel in 3
acetic acid, and bring destained gel to
Proteomics (Invitrogen SilverQuest
Protocol) Visualize down to 3 ng of
protein. Protocol listed below is for one
mini-gel, 1.0 mm thick, for large gels double all
solution volumes while keeping the same
incubation time. Please do not use the fast
protocol listed in the SilverQuest manual or the
destainer. Development times between 5 and
6 minutes yield the best mass spectrometry
results. Longer development time will result
in burning of the protein bands, as seen here
(over developed gel). Burning decrease peptide
recovery which decreases the opportunity for
positive protein identification.
Protocol for In-Solution
Reduction/Alkylation(prior to loading the
gel) Reduction 1. Protein is lyophilized,
precipitated or dissolved in 10 µL pH 7.5-8.5
buffer. 2. Resuspend protein in 20 µL 0.25
Zwittergent 3-16 (Calbiochem) in 200 mM ammonium
bicarbonate. 3. Add 1 µL of 50 mM DTT
(dithiothreitol) in water. Incubate 15 min at
60 oC. Let cool. (Note 50mM DTT 0.77
g/mL) Alkylation 1. Add 5 µL of 22 mM
iodoacetamide in water. Incubate 25 min at room
temperature in the dark. (Note 22 mM
iodoacetamide 0.004 g/mL) 2. Make a 110
dilution of the DTT solution from step 3 above
and add 1.4 µL to the sample. Incubate 25 min
at room temperature in the dark.
5
Protocol for Trypsin Digestion in Solution 1.
Calculate what a 150 EnzymeSubstrate ratio
would be. 2. Reconstitute Promega trypsin in 25mM
Ammonium Bicarbonate immediately prior to
adding to samples. 3. Add calculated amount of
trypsin solution to each sample. 4. Vortex/spin
down. 5. Incubate for 4hr in sealed sample tubes,
at 37C with rotation (400rpm). Protocol for
SYPRO RUBY STAIN The following procedure uses
a volume of 100mL per gel. A maximum of 5 gels
should be allowed in a single tray. All steps can
be performed at room temperature. Gently agitate
the trays on a rotary shaker at low speed during
all steps. Staining trays should be very clean
and it is best to rinse with ethanol prior
to use. Molecular Probes recommends the use of
polypropylene dishes or polyvinyl chloride
photographic staining trays for staining as they
adsorb the least amount of dye. SYPRO RUBY IS A
FLUORESCENT STAIN THAT MUST BE PROTECTED FROM
LIGHT BEFORE, DURING, AND AFTER STAINING STEPS.
TAKE CARE TO KEEP GELS COVERED DURING THE
STAINING PROCESS. 1. After electrophoresis, fix
2D gels in 40 methanol, 10 acetic acid for
60minutes. Fixing is not required for 1D
SDS-PAGE. 2. Incubate gels in SYPRO Ruby Protein
gel stain for 4 hours to overnight.
Continuous, gentle agitation should be maintained
during all staining and washing steps. For
optimum results, stain gels in polypropylene,
polycarbonate or polyvinyl chloride trays glass
trays will interfere with the staining
procedure. (Gels can be stained overnight for
convenience.) 3. Wash gels in 10 methanol, 7
acetic acid for 1hour. Repeat. 4. Gels are ready
for imaging. To improve the chance of
success, Invitrogen/Novex Pre-Cast Tris/glycine
SDS-PAGE gels and recommended staining procedures
MUST be used.
6
Protocol IV In gel digestion (??, 12/16/2003) 1.
Gel excision(3 parti. In coating tube) 2.
Destaining (coomassi) a.5-10min in DW
200ul(multi vortexing) b. 5-10min in 40
MeOH(??? ??? ??) c. 5-10min in ACN
(destaining ?? ?? b?? ??(2-3?) (silver) a.
wash the gel with water b. add 50ul of
Destainer A and 50ul of Destainer B(silver c.
mix throughly and incubate for 15min at room
tmp.(the gel pieces will slowly settle to the
bottom) d. remove the supernatant using a
clean pipette tip. e. add 200ul of water and
mix. Incubate for 10min at RT f. repeat steps
d-e at least two times 3. washing a. DW
washing b. 5-10min in 50mM ABC(pH8.5 ??)
c. 5-10min in (50mM ABCACN11) d. 5-10min
in ACN 4. Reduction and alkylation a. Swell,
in 50ul of reduction buffer(10mM DTT in 50mM
ABC), for 30min at 56?. b. Spin down,
discard, shrink with 50ul of ACN c. Replace
ACN with 50ul of alkylation buffer(55mM
iodoacetamide in 50mM ABC), incubate 20min at RT
in the dark. Discard IAM solution. d.
Wash with 100ul of 50mM ABC for 10min and add
100ul of ACN for 5min and discard. e. Wash
with 100ul of ACN for 5min f. Dry(speed
vac. 30min) 5. Trypsin digestion stock
trypsin(the modified porcine trypsin sequencing
grade,Promega) 20ug 200ul of 50mM
ABC,20ul ? aliquoting ?? ?? ??(0.1ug/ul),
mixture solution 20 ul of stock 140 ul of
50mM ABC containing 5.7mM CaCl2   17(v/v)),
2000ng/160ul 12.5ng/ul)
10 ul of stock 70ul of 50mM ABC
containing 5.7mM CaCl2   17(v/v)), 1000ng/80ul
12.5ng/ul a. put 20ul of mixture solution
buffer(12.5ng/ul trypsin in 50mM ABC containing
5mM CaCl2) to the tube in ice for 1-2
hours. (??? mixture solution (sample?
1(control)) x20ul)) b. Remove the
solution(??trypsin), add 50mM ABC(10-20ul, ?????)
and put it in an incubator(37?) for
overnight or 5hrs c. directly MALDI d.
extract 1) add 10 to 15ulof 25mM ABC,
vortex brifely, incubate at rt for 10min, recover
after a brief spin. 2) add ACN (1 to 2
times vol of gel), incubate at 37? for 15min with
agitation. Spin down and collect. 3) Add 40
to 50 ul of 5 FA, incubate at 37? for 15min with
agitation. Spin down and collect. 4) add
ACN (1 to 2 times vol of gel), incubate at 37?
for 15min with agitation. Spin down and collect.
e. pool, lyophilize and dissolve in 0.1 TFA(or
5FA)
In solution digestion (?? 12/16/2003 , Anal.
Chem. 1996, 68, 1-8) 1. Sample volume 10ul ?? 2.
DTT 10mM in 100mM ABC(pH 8.0) at 56 ? for 1h.(vol
50ul of 10mM DTT in 100mM ABC) 3. Iodoacetamide
55mM in 100mM ABC at RT for 30min.( vol 50 ul of
110mM IAM in 100mM ABC) ( ???? ?? ??, No
Light!) 4. trypsin substrate 130 (w/w),
stock trypsin (0.1ug/ul) 37? for overnight(more
than 5hrs) 5. Stopped by freezing the samples at
-20 ? 6. lyophilized and dissolved in 0.1 TFA(or
5FA)
7
Tips for Sample Preparation Gel 1. It is
strongly recommended that you use coomassie stain
rather than silver. If you can not visualize
bands with coomassie try to scale up your
isolation to increase the amount of
protein until you get to coomassie detectable
limits. 2. If you can not reach coommassie
stainable levels you must use a mass spec
compatible silver stain protocol. Only use
methanol and acetic acid during the fixing step.
Do not use any solutions containing
formaldehyde or glutaraldehyde to fix the gel.
Reference Shevchenko et al. (1996) Analytical
Chemistry, 68850-858 for more details. 3. If
possible run multiple lanes and combine several
bands together. 4. Use 1.0 to 1.5mm gels. 5.
Only stain the gel long enough (usually only a
few minutes) to detect the bands of interest. 6.
Take a picture of the gel and submit it
(photocopy or electronic) along with samples. 7.
Excise gel band(s) with as little excess empty
gel as possible. 8. Place the gel band(s) into a
micro centrifuge tube with some ddwater. Sample
quantityThe resolution and sensitivity of
Micromass Q-Tof 2 is superior. It appears that
samples at 200 fetomoles may be enough for a
protein ID. However, to achieve high accuracy, we
would like to have sample at 1 -2 pmoles. For the
SDS-gel sample, if a band is visible with
Coomassie blue stain (detection limit, 100 ng),
there is probably enough material present for
protein ID. SolventUsing the appropriate
solvents is very important for mass spectrometry.
Volatile solvents such as methanol and
acetonitrile are good for mass spectrometry.
Avoid using DMSO, DMF, and large polar solvent.
Always use spectroscopic grade solvent to prepare
the sample. Mixture and ImpuritiesPure samples
always provide more satisfactory data. Multiple
component samples will compete for protons in the
ionization process. The components with the
highest concentration will give strongest signal
and the components with low concentration will
give weak signals. In this case, the weak signals
(desired peaks) will be suppressed. However, this
can be circumvented when liquid chromatography is
configured with the electro-spray mass
spectrometry to separate components. Salts and
BuffersSalts and buffers are, in general,
detrimental to MS analysis. Salts normally form
adduct peaks which compete with the molecular ion
peaks and broaden the overall signal (especially
for protein analysis). ESI-MS is sensitive to
salts and buffers resulting in signal
suppression. Less than 1mM salts and buffers is
recommended although higher salt concentrations
may be tolerated. Ammonium acetate usually do not
affect the signal greatly below 20 mM. However,
sodium and potassium can be a real problem above
10mM. Protein/Peptide sample handling Always
wear gloves (powderless, rinsed with water and
ethanol before use) to eliminate contamination by
keratins, etc. Try to work as cleanly as
possible, because contamination with other
proteins could prevent identification of the
interesting protein. The most frequent
contaminants are BSA and human keratin. The
keratin comes from dust, small hairs and
fingerprints. Even a small hair contains
overwhelming amounts of keratin compared to the
amount of your sample. Use clean dishes for gel
casting as well as staining. Use fresh high
purity reagents and water. Contaminants from
buffers, detergents, guanidine HCl, urea, etc.
may affect your protein ID. Always use
siliconized polypropylene tubes as well as
low-retention tips to minimize protein loss by
adsorption to tube walls. Do not use glass tubes.
Free acrylamide may react with the amino groups
on proteins during polyacrylamide gel
electrophoresis. To avoid this, either buy
pre-cast gels for your electrophoresis system or
cast the separation gel 24 hrs. before using and
allow overnight polymerization. Excise the
bands, be sure to use extremely clean surfaces
and new razor blades or scalpels. Ideally this
should be done in a laminar flow hood (tissue
culture type) to minimize the possibility of any
dust, hair, flakes of skin, or other forms of
dirt.
8
Protein Identification Overview Proteins may be
identified by several related methods and
platforms. The steps involved are similar in
purpose regardless of the application employed.
The basic steps are protein separation, chemical
and/or proteolytic digestion of any protein or
peptide that is larger than 4 kDa, mass-to-charge
ratio measurements on the fragments produced in
the digestion by mass spectrometry (MS) and
database matching of fragment masses. Protein
separation is generally achieved through a series
of procedures. Initially a subset of cellular
protein is prepared which could include soluble
fraction, membrane fraction, mitochondrial
preparations, nuclear preparations etc. For
proteins in solution such as a cellular fraction,
serum, CSF, urine etc., additional separations
should be performed based on some physical
property such as isoelectric point (pI),
molecular weight, polarity, immunoaffinity, HPLC,
MDLC, etc. Finally, some combination of
separation strategies must yield a protein
fraction with a complexity of 1-3 protein species
which may be able to be identified by PMF or a
peptide which can be analyzed by MS/MS for amino
acid content or sequence. Gels can be stained
with Coomassie blue, silver stain, or Sypro Ruby.
The latter is a fluorescent stain with
sensitivity equal to that of silver stain, with
the advantage of a quantitative response over
three orders of magnitude. In addition, this
stain does not interfere with subsequent mass
spectrometric characterization of proteins and
peptides. 1D gel preparation for trypsin
digestion A 1D PAGE gel of appropriate
acrylamide (based on molecular weight range of
interest) is electrophoresed. The gel is stained
using an MS-friendly stain such as BioRad
Bio-Safe Coommassie Blue with a 2 h destain in DI
water, or Invitrogen Silver Quest Kit stain when
more sensitivity is required. Note that
fixatives (e.g., glutaraldehyde, formaldehyde,
high concentrations and long presence of
methanol) must be avoided. The band of interest
is then picked from the gel using a sterile,
disposable gel-plugging tip 1mm in diameter. The
gel band can be stored in up to 1 aqueous acetic
acid at 4C for up to 48 h prior to trypsin
digestion. Alternatively, the gel plugs may be
incubated with 11 (v/v) MeOH 50 mM NH4HCO3 and
dehydrated in acetonitrile, then dried in a
SpeedVac for storage at -80C for up to 3
months. 2D gel preparation for tryptic
digestionThe GPCL will perform traditional 2D
and difference (DiGE) gel electrophoresis upon
request. The GPCL staff will train and assist in
data analysis at an hourly rate beyond an initial
2 h training at no charge. Once a list of protein
spots to be picked has been chosen, the gel is
re-imaged, gel plugs are excised from the gel
and, depending on sample number and picking
method, placed into tubes or microtiter plates.
For DiGE the gel plugs may only be excised using
an automated picker into microtiter. For
traditional 2D gels, the gel plugs may be excised
manually or with an automated picker. If
traditional methods are used, the gel must be
stained with an MS-friendly stain (see above). An
investigator may also provide gel plugs, which
the Core will process directly. Gel and Gel plug
storageGel pieces for trypsin digestion and
protein ID should be processed as soon as
possible after the gel has been electrophoresed.
A gel or gel plug may be stored up to 48 h in 1
acetic acid prior to digestion. Beyond 48 h, some
loss of protein coverage will be observed and may
reduce the confidence in MS-based protein ID.
Alternatively, the gel plugs may be prepared for
long term storage as detailed above. (Up to two
gel plugs from the same protein spot/band should
be incubated in 11 (v/v) MeOH 50mM NH4HCO3
twice (30 min each incubation) at room
temperature. For a 1 mm diameter plug cut from a
1 mm thickness PAGE gel, 100 µl should be used.
Buffers are then carefully removed, the plug is
dehydrated in 50 µl of acetonitrile for 15 min,
then placed in a SpeedVac at room temperature
until dry (about 45 min). Following dehydration,
the gel plugs may be stored at -80C for up to 3
months the gel plug should have an opaque/white
appearance.) Peptide Mass Fingerprinting
Trypsin, the quantity of which is determined
based on the protein load in the sample, is added
to the gel plug. This mixture is incubated at
42C for 2 h, then extracted with in 11
acetonitrileH2O containing 0.3 aqueous
trifluoroacetic acid (TFA). The supernatant is
dried in a SpeedVac and resuspended with in the
same solvent, mixed with MALDI matrix and
immediately spotted onto the MALDI target for MS
and MS/MS analysis. The mass spectra for each
tryptic digest will be measured. The analysis
will be performed with the ABI 4700 instrument in
reflectron mode. The resolution of the
instrument, which has a 3.75 m ion flight path,
at 2.5 kDa is gt 12,000, and mass accuracies of
/- 5 ppm with internal standardization and /-
25 ppm with external standardization are
routinely achieved. This allows for ID of
proteins within gel spots or bands containing 1-4
proteins.
9








Q How much protein coverage does a typical
identification yield?A Coverage is very much
dependent on the amount of protein present and
also the nature of the protein itself. For
abundant proteins coverage can be high as 70-80
whereas low abundance proteins can be identified
from as little as one or two peptides (with
sequence information). Q How much material do I
need for the protein analysis?A Although the
sensitivity of our mass spectrometers is very
high, it is always prudent to send as much
protein as possible. We can identify (or obtain
sequence) from anything that can be seen by
coomassie or SyproÔ Ruby protein stain. Silver is
more problematic due to the nature of the stain
and its interaction with the protein but we
obtain signal from over 90 of samples. Listed
below are some arbitrary numbers for you to get
started.Protein detection limits for silver and
Coomassie staining were 1 and 100ng
respectively. How much sample should I submit?
You should submit at least 0.5 ml of the sample
with a minimum concentration of 100 mM.  (for
solid samples 150 pM or 0.1 ug) What are the
other requirements the sample should meet? 1.
For MALDI-TOF analysis, the sample should be
non-volatile. 2. For ESI-MS analysis, sample
should be soluble in polar solvents. 3. For both
MALDI and ESI, inorganic buffers and other
impurities should be minimized or eliminated.
The samples should not be radioactive, unusually
toxic or require a biohazard facility for
analysis. What type of solvent shall I use for
the sample preparation? The solvent used for
dissolution should be volatile. If the sample is
a pure stable solid, please provide information
regarding its solubility. For either solids or
solutions buffers and electrolytes should be
minimal or absent. Do I have to submit any
other material with the sample? Yes, you will
have to submit the analysis request form which is
available on-line or you can download it and send
it with the sample. Also with this form please
submit any additional information on a separate
sheet of paper such as the suspected molecular
structure and any mass spectrometry data.  Sample
history (isolation, purification, synthesis) and
information regarding the objective of the
measurement and the overall experiment is often
very helpful in designing the MS measurement and
understanding signals from unexpected impurities.
Which method shall I use for my sample
analysis? The choice of analytical MS methods
will be made by the facility staff based on the
information provided about the nature of the
sample, the information needed and the type of
sample to be analyzed.  Submitters may also
indicate a preference or request a specific type
of analysis.
10

• Mass Data processing
• Database search Each acquired MS, MS-MS spectrum
  is compared with theoretical spectra obtained
  from
• a sequence database.
  
• a. database search tools Profound, Mascot,
  Sequest (different scoring function)
• b. Databases protein sequence database(NCBInr,
  SwissProt..), genomic database, EST database
• (only enables identification of those
  peptides that are present in the searched
  sequence databases)
• c. To determine whether best match assignment
  is correct, need manual validation of database
  search results
• (simplicity of the search tools, uncertainity
  of charge state, low quality of MS-MS spectra,
  not present in the
• database)
• 2. De novo sequencing
• a. De Novo sequencing tools
• Lutefisk, De Novo, PEAKS, PepSeq program of
  MassLynx(micromass) Analyst Q(Q trap, Q-star)
• De Novo sequencing(4700)
• b. Derived peptide sequences can then be searched
  against a protein sequence database using BLAST-
  or
• FASTA-type sequence similarity search
  algorithms to infer the identities of their
  corresponding proteins.
• c. manual validation time consuming, consistent
  and objective evaluation of the data, requires
  significant
• expertise

11

• Database searching was done using the Mascot
  search engine.
• A combination of computer scoring and human
  criteria were employed in the screening of the
  data.
• First the data was searched against the RefSeq
  database with tryptic constraints and a base list
  of proteins was generated on
• which further analysis was performed.
• An initial list of proteins was generated by the
  following procedure
• Only proteins containing at least 1 unique
  peptide (we refer to peptide as being unique for
  specific protein, if the sequence has not
  previously been used to assign to a different
  protein) with a peptide Mascot score greater than
  20 were considered.
• b) The highest scoring peptide for each of the
  protein entries generated in a) was manually
  inspected and interpreted to confirm the identity
  of the peptide. If the spectrum could match a
  different sequence better than the assigned one,
  if it had poor ion statistics, or if no other
  good spectra pointed to the same protein, the hit
  was discarded. In addition, the inspected peptide
  match was required to have a length of at least 8
  amino acids and to have a sequence tag of at
  least three amino acids preferably a good y-ion
  series. Also, the y1 and a2-b2 pair, if present,
  had to be consistent with the identified peptide
  sequence. If the sequence tag was not composed of
  y-ions and if no other peptides matched the same
  protein, the hit was discarded unless the
  spectrum was of good quality and most peaks could
  be explained.
• c) If a protein has multiple isoforms or has
  multiple entries in the databases, we only
  specify the major form of the protein unless a
  specific peptide points to a region of the
  protein, which exists only in one of the
  isoforms.
• d) If multiple peptides that matched the same
  protein are not from the same vicinity on the gel
  (more than two gel bands away), then extra care
  is taken to confirm those entries.
• In order to identify proteins, which are not
  present in the RefSeq database, the peak list
  file was searched against the nr database at NCBI
  and the results compared with the list generated
  by searching the RefSeq database.
• New entries retrieved from the nr database were
  tested against the same criteria as described
  above for identification purposes.
• If a spectrum, that had been used to confirm an
  entry in the RefSeq derived dataset, fitted an
  entry better in the search against the nr
  database, the original hit was removed (e.g. a
  different splice variant).

12
Guidelines in Publication of Peptide and Protein
Identification Data (Mol Cell Proteomics. 2004,
531-)
1. The following supporting information . The
method and/or program used to create the peak
list from raw data and the parameters used in
the creation of this peak list, particularly any
that might affect the quality of the subsequent
database search. Examples include whether
smoothing was applied any signal-to noise
criteria the percentage peak height at which
centroids were calculated and whether charge
states were calculated and peaks de-isotoped. .
The name and version of the program(s) used for
database searching and specific parameters used
for its (their) operation. Examples include
precursor-ion mass accuracy fragment-ion mass
accuracy modifications allowed for any missed
cleavages enzyme specified or not etc. . Scores
used to interpret MS/MS data and thresholds and
values specific to judging certainty of
identification, whether any statistical analysis
was applied to validate the results, and a
description of how applied. . The name and
version of sequence database used the count of
number of protein entries in it at the time
searched. 2. Information regarding the sequence
coverage observed for each protein should be
provided either in the manuscript or in the
supplementary materials. The authors are
encouraged to provide a table that lists for each
protein the sequences of all identified peptides.
At the minimum, the total number of peptides
belonging to each protein must be explicitly
stated either in the text or in tables presented.
To compute this number, different forms of the
same peptide are to be counted as only a single
peptide. For example, if the same peptide is
identified in both 2 and 3 charge forms, the
number of interpreted spectra equals 2, but
the count of identified peptides that count
toward the protein sequence coverage measure is
only 1. Similarly, if multiple forms of a peptide
are identified that arise through common
sample-handling artifacts (e.g., oxidation of Met
and deamidation of Asn), then the count of
peptides identified again equals 1. The total
number of MS/MS-interpreted spectra assigned to
peptides corresponding to each protein can also
be provided, but this should not be confused with
the protein sequence coverage measure described
above. 3. Protein assignments based on
single-peptide assignments must include
additional information in the table(s).
Specifically, we require authors to show 1) the
sequence of the peptide used to make each such
assignment, together with the amino acids N- and
C-terminal to that peptides sequence, 2) the
precursor mass and charge (not just m/z)
observed, and 3) the scores for this peptide (in
the case of multiple MS/MS spectra assigned the
same peptide, this information should be provided
only for the best assignment). As noted in
guideline 2, assignments of the same peptide to
multiple MS/MS spectra of the same charge form
are to be counted as single-peptide
assignment. 4. In cases where biological
conclusions are based on observation of a single
peptide matching to a protein or a
posttranslationally modified form of that
protein, then this identification must be
supported by inclusion(in the main body or in
supplemental materials) of the corresponding
MS/MS spectrum appropriately labeled.
13
5. Peptide mass fingerprint data will continue to
be accepted for peptide identification, but the
standard of acceptability will be more stringent
than currently allowed. In addition to listing
the number of masses matched to the identified
protein, authors should also state the number of
masses not matched in the spectrum and the
sequence coverage observed. They must describe
the parameters and thresholds used to analyze the
data (see guideline 1, above), including mass
accuracy, resolution, means of calibrating each
spectrum, and exclusion of known contaminant ions
(keratin, etc.). Authors are encouraged to use
and provide the results of scoring schemes that
provide a measure of identification certainty, or
perform some measure of false-positive rate. 6.
Essentially the same protein appears in many
cases under different names and accession numbers
in the database. When matching peptides to
members of such a family, it is the authors
responsibility to demonstrate that they are aware
of the problem and have taken reasonable measures
to eliminate redundancy. In cases where a
single-protein member of a multi-protein family
has been singled out, the authors should explain
how the other members of the group were ruled
out, if at all. In addition, sometimes proteins
are identified from a different species than the
studied one. For example, mouse or human protein
in a hamster study. If such a protein is
included, it also has to be mentioned and
justified. 7. MCP strongly encourages (but does
not at present require) the submission of all
MS/MS spectra mentioned in the paper as
supplemental material. We will accept dta, pkl,
and mgf files. Because technical aspects of
storing large repositories of raw mass
spectrometric data has yet to be worked out, MCP
cannot at present accept any such data. However,
authors are encouraged to provide access to raw
MS data using other means, including group
websites and public repositories as they emerge.