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