Protein digestion and
peptide mass fingerprint
(PMF) analysis
Yuanming Luo
Institute of Microbiology,CAS
Protein digestion
In gel digestion
On-membrane digestion
In solution digestion
Two main approaches for
digesting gel-separated proteins
Protein blotting onto immobilizing
membranes followed by on-membrane
digestion (simpson et al., 1989;
Gevaert and Vandekerckhove 2000)
Digesting proteins directly in the
gel matrix and extracting the
peptides (Ward et al., 1990)
Note:
The overall recoveries of peptides
from in-gel digestion methods are
significantly greater than that from
on-membrane digestion strategies.
In-gel digestion might fail when the
visualized protein is below~10mg of
protein/cm2 (need a further
concentration of weakly stained CBB
gel spots (membrane spots) with
electrophoresis in a new gel).
In gel digestion
1. Excise the protein gel spots of
interest and place in microfuge tube.
2. Remove excess CBB by washing twice
with 1 ml of either 0.1 M NH4HCO3,
50% acetonitrile.
3. Dry each gel piece completely by
speed Vac. The gel piece should not
stick to the walls of the EP tube
when completely dry.
In gel digestion (continued)
4. Rehydrate the gel piece by adding
10ul of digestion buffer, containing
0.5ug of the appropriate protease,
directly onto the dried gel piece.
5. Store the gel pieces at 4℃ for
over 45 min until the solution has
been absorbed
6. If necessary, repeat above two
steps to allow the gel pieces to
fully swell.
In gel digestion (continued)
7. Add 20 ul of digestion buffer
without protease to fully immerse the
gel piece.
8. Incubate for 12-16 hours at 37 ℃.
9. Carefully remove the digestion
buffer (now called the extract), and
place it into a clean microfuge tube.
The digestion buffer contains > 80% of the extractable
peptides
10. Add 200 ul of 5 %TFA/50%
acetonitrile to the gel piece.
In gel digestion (continued)
11. Incubate the tube with the gel
piece for 1 hour at room
temparature(37 ℃).
12. Carefully remove the extract away
from the gel piece and combine it
with previous extract from step 9.
Repeat step 10,11,12 once again.
Concentrate the pooled extracts by
speed Vac. Store the peptide extracts
at -20 ℃ for future mass
spectrometric analysis.
In gel digestion of silverstained gels
Most of the steps of in gel digestion
for silver-stained gels are the same
except for the destaining solution
which was prepared by mixing 30 mM
potassium ferricyanide with 100 mM
sodium thiosulfate (1:1 v/v).
On-membrane proteolytic
digestion of electroblotted
proteins
Proposed membranes are nitrocellulose
membranes, because the hydrophobic
surface of PVDF limits the recovery
of peptide fragments.
Proposed dyes are Amido Black and
Ponceau S, which are compatible with
proteolytic digestion, peptide
extraction from membrane and
subsequent RP-HPLC analysis of
peptides.
Electroblotting of staining the
protein
1. electroblot the proteins from the
gel onto a nitrocellulose (for proteins
that are difficult to transfer, add up to 0.005%
SDS)
2. stain the membrane with either
Amido Black or Ponceau S.

E.g., for staining with Amido
Black
 A. immerse the nitrocellulose membrane in
0.1% Amido Black 10B for 1-3 minutes
 B. Rapidly destain with several washes of
Electroblotting of staining the
protein (continued)
C. Rinse the destained blots
thoroughly with deionized H2O to
remove any excess acetic acid.
D. cut out the stained protein band
(or for 2D gel spots, up to 40 spots
from identical gels may be required)
and transfer these bands to 1.5-ml
microfuge tubes for immediate
processing (begin with step 3) or for
storage at -20℃.
For staining with Ponceau S
 A. Immerse the nitrocellulose membrane in
0.1% Ponceau S for 1 minute.
 B. Gently agitate the blot for 1-3 min in
1% acetic acid to remove excess stain.
 C. Cut out the protein bands of interest
and transfer them to microfuge tubes.
 D.Destain the protein bands by washing the
membrane pieces with 200 mM NaOH for 1-2
min.
 E. Wash the membrane pieces with deionized
H2O and process them immediately or store
them wet at -20℃(avoid excessive
drying).
Digestion of the membrane-bound
proteins
3. Add 1.2 ml of 0.5% (w/v) PVP-40
(which is used to prevent absorption
of the protease to the nitrocellulose
during digestion) in 100 mM acetic
acid to each tube.
4. Incubate the tube for 30 min at 37
℃.
5. Centrifuge the tube at ~1000g for
5 min.
6. Remove the supernatant solution
 7. Add ~1ml of H2O to the tube.(It
is
essential to remove excess PVP-40 before peptide
mapping because of the strong UV absorbance of
this detergent. Moreover, breakdown products of
PVP-40 produce major contaminant peaks in ESI-MS)




8. Votex the tube for 5 seconds.
9. repeat step 5 and 6.
10. repeat steps 7-9 five more times.
11. cut the nitrocellulose strips
into small pieces (~1x1mm) and place
them in a fresh tube (0.5- or a 0.2ml tube).
Digestion of the membrane-bound
protein (continued)
 12. Add the minimal quantity of
digestion buffer (10-20ml) to
submerge the nitrocellulose pieces.
 13. After digestion, tryptically 16
hours or overnight at 37 ℃, load the
total reaction mixture onto an
appropriate RP-HPLC column for
peptide fractionation (or store the
peptide mixture at -20 ℃ until use).
In soltion
digestion
Mass spectrometry involved in
proteomics
 Matrix-associated laser desorption ionization
time of flight mass spectrometry (MALDI-TOFMS)
 Electrospray ionization (ESI) ion trap mass
spetrometry
 Surface-enhanced laser desorption ionization
(SELDI) time of flight mass spetrometry
Linear and reflectron MALDI-TOFMS
Linear MALDI-TOF-MS (lower accuracy)
Reflectron MALDI-TOF-MS: (1) higher
accuracy, (2) Post-source decay (PSD),
(3) delayed extraction
Mass spectrometry terms
Mass to charge (m/Z): Mass
spectrometers measure the mass-tocharge values of molecular ions.
Resolution: resolution can be defined
as the ability to separate and
measure the masses of ions of similar,
but not identical, molecular mass.
Signal to noise:
Monoisotopic mass versus average mass:
What is the difference between
monoisotopic and average peptide mass?
 As shown in the table, below the atoms that make up the
naturally occurring amino acids found in proteins are not
isotopically
pure.
Natural
Abundance of Isotopes Commonly Found in Proteins
Atom
Most Abundant Isotope
Next Most Abundant Isotope
Carbon
12C
98.9%
13C
1.11%
Nitrogen
14N
99.6%
15N
0.366%
Oxygen
16O
99.8%
18O
0.204%
Sulfur
32S
95.0%
34S
4.22%
Deisotoped MALDI spectrum
n
MS
Interpreting of the
spectra of peptides (AGFI)
Singly-charged peptide
fragmentation
Reaction products: Proton on
carbonyl (case 1)
Reaction products: Proton on
amine (case 2)
Doubly-charged peptide
fragmentation
Reaction products: Proton on
carbonyl (case 1)
Reaction products: Proton on
amine (case 2)
Where is the b1 ion? Why is the
a2/b2 big?
Peptide mass fingerprint
(PMF) analysis by MALDITOF-MS
MALDI difficulties
Salts and other contaminants
Selection of matrix
Sample itself
Crystallization
Calibration (external or
internal cablibration)
Accuracy (ppm)
Resolution
Amount of protease
Dissolve the peptide mixture in
0.1%TFA.
Desalt by ZipTip C18 microcolumn
(optional).
Directly elute the peptide mixture
with -cyano-4-hydroxy cinnamic acid
(CHCA) in 70% acetonitrile and spot
the peptide mixture on sample plate.
The peptide mixtures cocrystallize
with matrix on sample plate
Input of instrumental method and
parameter setup
Calibration of spectrometer by
external calibration
Crude spectrum
Tuning of parameters
Internal calibration of PMF spectra
Data processing for database search
Peptide and protein standards
 Angiotensin II (human)
Substance P (human)
Insulin (bovine)
5733.6
Cytochrom c (equine)
12,360.1
RNase A (bovine)
13,682.2
Apo-Myoglobin (equine)
16,951.5
MW: 1046.2
MW: 1347.7
MW:
MW:
MW:
MW:
Manually spot samples onto
sample plate
Recrystallization methods for MALDI
matrices
Method 1:
Re-crystallization in 70% MeCN/30%
water.
1.Heat a saturated solution of the
matrix in 70% MeCN/30% water until
boiling.
2.Carefully boil until solid dissolves
completely.
3.Cool to room temp, then on ice precipitate should form.
4.Filter precipitate.
Recrystallization methods for MALDI
matrices
 Method 2:
 Protocol for re-crystallization of alphacyano-4-hyrdoxy-cinnamic acid
1.To 100mg of alpha-cyano-4-hydroxy-cinnamic
acid, add 10ml of water
2.Add ammonium hydroxide until most of acid
dissolves.
3.Slowly add concentrated HCl to the
solution until a large amount of
the acid has precipitated (about pH 2).
4.Remove the precipitate by centrifugation
or filtering.
5.Wash the precipitate several times with
0.1M HCl.
Calibration
Used for
calibration
It would be extremely difficult to measure a monoisotopic mass for BSA. In practice,
most instruments report monoisotopic molecular weights up to a certain cut-off point.
Above this cut-off, isotopic envelopes are centroided as a whole to provide average
mass values.
Control the
sample plate
CHCA
Parameters setup of PMF
Instrument：Voyager DETM PRO
BiospectrometryTM workstation,
Parameters： matrix:α-Cyano-4hydroxycinnamic acid(CHCA)
(1)delayed extraction, 150 ns、
(2)reflector mode、(3)positive;
(3)acceleration voltage: 20000 V；
(4)Grid voltage75%；(5)Guide wire:
0.02%；(5)mass range: 800-3500 Da。
PMF containing isotope
peaks
monoisotope spectrum
PMF spectrum of annexin I
Voyager Spec #1 MC=>BC=>NF0.7[BP = 1746.9, 43815]
1746.9067
100
90
1011.5399
4.4E+4
1543.8638
80
1816.9505
1651.8949
70
1945.0510
1262.5952
% In ten sity
60
1092.6082
1725.8451
50
1064.5430
1523.8085
40
2149.9616
908.4443
30
1803.7894
912.5189
1749.9014
20
10
0
799.0
855.4336
936.5116
2345.1750
1948.0341
1640.8004
2723.3341
2164.0483
1213.5503
1339.2
1387.7486
1823.8325
1879.4
2419.6
Mass (m/z)
2959.8
0
3500.0
Mass list of annexin I
(monoisotope)
855.433556
908.444342
912.518921
936.511625
966.487904
982.482524
1011.539929
1064.543038
1092.608231
1213.550297
1262.595173
1523.808451
1543.863778
1640.800435
1649.789297
1651.894876
1724.838185
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1746.906727
1768.874981
1803.789401
1807.994050
1815.963445
1816.950528
1820.832451
1903.003019
1944.068873
1945.050987
2132.706314
2148.959052
2163.051584
2203.054533
2344.153915
2722.314675
Database search by MASCOT
Key points for PMF
 Sample preparation
 Matrix selection and ratio of sample to
matrix
 Keep the matrix dry
 Sample properties
 Internal calibration
 Database selection
 Mass accuracy (ppm)
 Resolution (FWHM) and S/N when tuning
Practical consideration
 -The final molar ratio sample/matrix is
about or around 1/5000.
 -Final concentration of the sample is from
1 to 10 pmol/ul
 -Our experience with MALDI point to a
dynamic range of 100 fmol/ul to few
hundreds pmol/ul
 -MALDI is relatively robust ionization
technique that tolerates the use of salts
and surfactants and buffers. Although it
is best to remove them for better
performance.
Theoretical digestion for PTM
identification analysis
Application of MALDI-TOF-MS
Molecular weight
determination
Peptide mass fingerprint
Disulfide bond
identification
C-terminus sequencing