1. Protein identification

2. Protein Identification
1. Peptide Mass Finger Printing (PMF)
from MS data
2. Database search using fragment ion masses
from MS/MS data
3. Sequence Tags

3. EXPERIMENTAL PROCEDURES IN PROTEOMICS
SEPARATION electrophoresis (1-D, 2-D)
chromatography (SEC, ion exchange, reversed phase)
DIGESTION chemical (BrCN)
enzymatic (trypsin,, Lys-C, Asp-C)
reduction (Di-Thio-Threitol, b-Mercapto-Ethanol)
alkylation (IodoAcAcid, IodoAcAmide, Vynil Pyridine)
SAMPLE CLEAN UP chromatography (reversed phase)
solid phase extraction (Zip Tip)
MALDI MS ANALYSIS
protein identification (peptide mass fingerprinting)
peptide structural information (post source decay)

4. Peptide Mass Fingerprinting
Peptide extraction
In situ digestion
MALDI-MS
Putative Candidates Score
1. Larval serum protein
2. Larval serum protein 2 precursor 493
3. CG7251-PA

5. Database Search with Peptide Mass Fingerprinting Results
GLSDGEWQLVLNWGKVEA
DIPGHGQEVLIRLFKGHP
ETLEKFDKFKHLKSEDEM
KASEDLKKHGATVLTALG
GILKKKGHHEAEIKPLAQ
SHATKHKIPVKYLEFISE
CIIQVLQSKHPGDFGADA
QGAMNKALELFRKDMASN
YKELGFQG
Protein sequence databank
GLSDGEWQLVLNWGK
SEDEMK
ASEDLK
YLEFISECIIQVLQSK
GHPETLEK
HGATVLTALGGILK
DMASNYK
ELGFQG
Generate all theoretical peptide
List all peptide masses
450.22
609.37
664.33
Theoretical Masses
Matches
ALB
GAD
Experimental
Masses
450.22
609.37
698.31
Results
GAD
High Score
ALB
Low Score

8. Effect of Mass Accuracy and Mass Tolerance on Peptide Mass Fingerprinting Search ResultsDa
Search m/z
N° of Hits

9. Effect of multiple Peptide Masses on Protein Identification by Mass Fingerprinting
Mass Tolerance Da
Search m/z
N° of Hits

13. ProFound Search - Result Summary
Protein Candidates for search C D8-294F31B
sequence searched
Ran Est’d Protein Information and
Probability % pl kDa ®
k Z Sequence Analyse Tools (T)
T° gi| |ref|NP
e zinc finger protein ®
[Homo sapiens]
e T° gi| |ref|XP
minichromosome maintenance ®
deficient (mis5, S. pombe) 6

14. ProFound Search - Input Summary

25. Mascot search output Mascot Search Results Significant hits:
ALBU_BOVIN (P02769) Serum albumin precursor (Allergen Bos d 6).
ALBU_CANFA (P49822) Serum albumin precursor (Allergen Can f 3).
VWF_PIG (Q28833) Von Willebrand factor precursor (vWF) (Fragment).
CIQ3_BOVIN (P58126) Voltage-gated potassium channel protein KQT-like 3
RYR2_RABIT (P30957) Ryanodine receptor 2 (Cardiac muscle-type ryanodin
K2CA_BOVIN (P04263) Keratin, type II cytoskeletal 68 kDa, component IA
ALFB_RABIT (P79226) Fructose-bisphosphate aldolase B (EC ) (Li
ALBU_BOVIN Mass: Total score: 711 Peptides matched: 12
(P02769) Serum albumin precursor (Allergen Bos d 6).
   Observed   Mr(expt)   Mr(calc)   Delta  Miss Score  Rank   Peptide  
15     461.80     921.58     921.48      0.10    0     55     1    AEFVEVTK  
19     501.80               0.01    0     31     1    LVVSTQTALA  
31     569.80               0.09    0     72     1    CCTESLVNR  
33     582.30              -0.04    0     76     1    LVNELTEFAK  
47     653.40               0.08    0     90     1    HLVDEPQNLIK  
58     722.40               0.15    0     87     1    YICDNQDTISSK  
60     739.80               0.07    0     43     1    ETYGDMADCCEK  
61     740.40              -0.00    0     61     1    LGEYGFQNALIVR 
64     751.90               0.18    0         1    EYEATLEECCAK  
74     547.30              -0.05    1     85     1    KVPQVSTPTLVEVSR  
97     627.70               0.16    0     34     1    RPCFSALTPDETYVPK  
98     636.70               0.16    0     42     1    LFTFHADICTLPDTEK

26. Effect of Mass Accuracy and Mass Tolerance on Peptide Mass Fingerprinting Search Results
Error: 300 ppm
Error: 100 ppm

32. 1 2 4 3 Automated LC- MS e MS/MS run 1553 (3+) 1553 (2+)
Chromatogram :
Total ion current vs. time 25 30 35 40 45 50 55 60 65 70 75 80
Time, min
0.00
5.00e4
1.00e5
421.68
653.32
464.17
582.25
722.28
501.73
507.75
740.41
595.29
700.34 1 2 3 4
Full scan : +TOF MS:
m/z, amu
400
450
500
550
600
650
700
750
800
850
900
200
518.82
536.80
662.76
531.47
1553 (2+)
1553 (3+)
MS/MS peptide 4 :
+TOF Product (662.8):
10.0
14.0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
m/z, amu
0.0
5.0
235.08
166.06
147.10
262.10
120.06
409.18
207.09
110.04
138.06

34. Sequence Tags from Peptide Fragmentation by MS/MS
One sequence tag includes four components:
peptide molecular weight (MW)
partial sequence (region 2)
molecular wt before partial sequence (region 1)
molecular wts after partial sequence (region 3) A V
I/L T
Peptide measured molecular wt =
Partial Sequence
- A-V-I/L-T-
381.1
region region region 3