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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 - PowerPoint PPT Presentation
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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
Trypsin
N KKK
K
KK
R
RRR
CN
C
K
KK
K
K
K R
R
R
RProtein
Tryptic peptide mixture. Masses measured by MS. Every peptide has a basic C-terminus.
A protein can be identified in a database by matching masses of a subset of the tryptic peptides against calculated values.
MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVS
PFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVW
EQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIK
SYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRE
SGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVL
LEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLE
PPPEHIPPPPRPPKRILEPHNGKCREFFPNHQWVKEETQEDKDCPIKEEK
GSPLNAAPYGIESMSQDTEVRSRVVGGSLRGAQAASPAKGEPSLPEKDED
HALSYWKPFLVNMCVATVLTAGAYLCYRFLFNSNT
intact protein
enzyme
peptide fragments
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 co-crystallize 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 Fingerprinting2D-Gel
In Gel Digestion
MS
848.11272.5492.6
883.22978.9
812.61432.33127.1996.8702.4164.92748.2
848.31272.7493.2882.62978.3364.1948.93128.8
Database
3514.22837.1263.9147.41429.7199.6142.3640.8
Is identical to
In Silico Digestion
“Spot removal”
901.0 1236.8 1572.6 1908.4 2244.2 2580.0
Mass (m/z)
0
3.1E+4
0
10
20
30
40
50
% In
tens
ity
Voyager Spec #1=>NR(2.00)[BP = 1567.2, 61395]
YLYEIAR
LGEYGFQNALIVR
RHPEYAVSVLLR
DAFLGSFLYEYEYSR
MPCTEDYLSLILNR
RPCFSALTPDETYVPK
RHPYFYAPELLYYANK
GLVLIAFSQYLQQCPFDEHVK
1. MALDI-TOF MS spectrum
901.0 1236.8 1572.6 1908.4 2244.2 2580.0
Mass (m/z)
0
3.1E+4
0
10
20
30
40
50
% In
tens
ity
Voyager Spec #1=>NR(2.00)[BP = 1567.2, 61395]
YLYEIAR
LGEYGFQNALIVR
RHPEYAVSVLLR
DAFLGSFLYEYEYSR
MPCTEDYLSLILNR
RPCFSALTPDETYVPK
RHPYFYAPELLYYANK
GLVLIAFSQYLQQCPFDEHVK
2. Data Search (data base sever: NCBI & Mascot)
3. Identification : Bovine Serum Albumin
• http://www.mass-spec.co.nz/submission/pmf_ss.pdf
• http://www.matrixscience.com/cgi/master_results.pl?file=../data/F981122.dat