Peptide Mass Fingerprinting
• Analytical technique for protein
identification (protein sequence)
• Unknown protein of interest cleaved into
peptide by protease
• Collection of peptides resulting from this
cleavage comprise a unique identifier of
the unknown protein
• Mass measured with MALDI-TOF
and ESI-TOF
• in silico compared to the genome
• Computer programs translate the known
genome of the organism into proteins
• Theoretically cut the proteins into peptides
with the same protease (ex.Trypsin: K or R)
• Calculate the absolute masses of the
peptides from each protein
• the masses of the peptides of the unknown
protein vs the theoretical peptide masses
of each protein encoded in the genome
• Results statistically analyzed to find the
best match
Peptide Mass Fingerprinting
• Advantage : only the masses of the
peptides have to be known
• Disadvantage :
- the protein sequence has to be
present in the database of interest
- most PMF algorithms assume the
peptides come from a single protein
Sample preparation
• SDS-PAGE →chemical modification
• Protein cleavage : trypsin, chemotrypsin,
or V8 protease
• Sample : protease = 50 : 1
• Peptides extracted with acetonitrile and
dried under vacuum.
• Peptides dissolved in a small amount of
distilled water
• Mass spectrometric analysis
• Sample generation (시료)
– Origin of sample
•
hypothesis, organism, environment,
preparation, paper citations
• Sample processing (시료 전처리)
– Gels (1D/2D), columns, other methods
• images, gel type and ranges, band/spot
coordinates
• stationary and mobile phases, flow rate,
temperature, fraction details
• Mass Spectrometry (질량 분석기)
• machine type, ion source, voltages
• In Silico analysis (데이터 분석)
• peak lists, database name + version, partial
sequence, search parameters, search hits,
accession numbers
In Gel Digestion & Mass Spectrometry
Trypsin Digest
Cut out 2D-Gel Spot
Protein
Peptides
Peptide Mass Fingerprinting
N
K
K
R
K
K
K
R K
R
Trypsin
K
Protein
N
K
K
K
R
R
R
R
C
K
K
R
C
Tryptic peptide mixture.
Masses measured by MS.
Every peptide has a basic Cterminus.
A protein can be identified in a database by matching masses of a
subset of the tryptic peptides against calculated values.
intact
protein
enzyme
peptide
fragments
MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVS
PFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVW
EQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIK
SYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRE
SGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVL
LEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLE
PPPEHIPPPPRPPKRILEPHNGKCREFFPNHQWVKEETQEDKDCPIKEEK
GSPLNAAPYGIESMSQDTEVRSRVVGGSLRGAQAASPAKGEPSLPEKDED
HALSYWKPFLVNMCVATVLTAGAYLCYRFLFNSNT
Mass spectrometric analysis
• Digested protein analyzed with different
types of mass spectrometers
; ESI-TOF or MALDI-TOF (allows higher
sample throughput and several proteins
analyzed in a single experiment)
• A small fraction of the peptide (usually 1
microliter or less) is pipetted onto a MALDI
target
• A chemical called a ‘matrix’ is added to
the peptide mix
• Matrix molecules required for the
desorption of the peptide molecules
• matrix : a chemical with the correct
properties that absorbs light, and so
energy, at the wavelength of the laser used
• Matrix and peptide molecules cocrystallize on the MALDI target
• Analyzed.
Computational analysis
• Peak list : the mass spectrometrical
analysis produces a list of molecular
weights
• Peptide masses compared to huge
databases which contain protein sequence
information
• MS-Fit, Mascot, Peptident and Profound
• Software programs cut all these proteins
into peptides with the same enzyme used
in the chemical cleavage (ex. trypsin)
MALDI-TOF MS Data acquisition
-Data Search [profound; Mascot; MS-Fit] -
• Absolute mass of all these peptides is then
theoretically calculated
• Comparison between the peak list of
measured peptide masses
vs all the masses from the calculated
peptides
• Results statistically analyzed
• Possible matches returned in a results
table
Peptide Mass Fingerprinting
2D-Gel
Database
“Spot removal”
In Silico Digestion
In Gel Digestion
MS
848.1
1272.5
492.6
883.2
2978.9
812.6
1432.3
3127.1
996.8
702.4
164.9
2748.2
Is identical to
848.3
1272.7
493.2
882.6
2978.3
364.1
948.9
3128.8
3514.2
2837.1
263.9
147.4
1429.7
199.6
142.3
640.8
1. MALDI-TOF MS spectrum
Voyager Spec #1=>NR(2.00)[BP = 1567.2, 61395]
3.1E+4
0
901.0
1236.8
1908.4
M ass (m/z )
RHPYFYAPELLYYANK
MPCTEDYLSLILNR
1572.6
GLVLIAFSQYLQQCPFDEHVK
10
RPCFSALTPDETYVPK
20
RHPEYAVSVLLR
LGEYGFQNALIVR
30
YLYEIAR
% Inte ns ity
40
DAFLGSFLYEYEYSR
50
2244.2
0
2580.0
2. Data Search (data base sever: NCBI & Mascot)
Voyager Spec #1=>NR(2.00)[BP = 1567.2, 61395]
3.1E+4
50
GLVLIAFSQYLQQCPFDEHVK
RHPYFYAPELLYYANK
RPCFSALTPDETYVPK
MPCTEDYLSLILNR
DAFLGSFLYEYEYSR
20
RHPEYAVSVLLR
LGEYGFQNALIVR
30
YLYEIAR
% Inte ns ity
40
10
0
901.0
1236.8
1572.6
1908.4
M ass (m/z )
3. Identification : Bovine Serum Albumin
2244.2
0
2580.0
• http://www.massspec.co.nz/submission/pmf_ss.pdf
• http://www.matrixscience.com/cgi/m
aster_results.pl?file=../data/F981122.
dat