Identifikasi protein dengan HRMS

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Mass Spectrometry (MS)
• Analytical technique for identification,confirmation, quantitation of
compound based on the measurement of mass to charge ratio ( m/z )

• key is to be charge of the compound can be identification ms

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MS/MS

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Workflow For Unknown Screening

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Workflow of Proteomics

LC-MS Data Data

Treatment Sample Pre Processing
Analysis Mining

Subcellular Separation; Digestion; Labeling; Peptide Fractionation; Desalting

DDA; MRM; DIA; Fragmentation: ETD/HCD/CID

Data Search: Sequest/MASCOT etc.

Functional Annotation/Pathways/Overrepresentation

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Bottom-up proteomics sebagai dasar pengembangan
analisis berbasis MS
• Semua makhluk hidup mempunyai hubungan
kekerabatan secara genetic
• Filogeni menunjukkan diversitas dan hubungan
kekerabatan.

• Filogeni dibuat dengan menggunakan urutan

DNA.

• Hubungan antara gen dengan spesies

(variabilitas) menjadi sentral dalam analisis halal
 Evolusi molekuler dari DNA ke protein
• 3 perbedaan nukeotida DNA pada
tiga kodon hanya menyebabkan
Gly Ala Ile Leu asp Arg perbedaan satu asam amino

-GGAGCCATATTAGATAGA- • 2 penggantian nucleotida

dikatakan sinonim dan satu
-GGAGCAATTTTTGATAGA- penggantian lain disebut non-
sinonim
Gly Ala Ile Phe asp Arg
Terdapat lebih satu kodon untuk
setiap asam amino.

Menemukan urutan protein spesifik

spesies menjadi semakin kompleks
disbanding urutan DNA, tetapi lebih
menjanjikan untuk pembeda
 Phase 1
Proteome mapping

Targeted
products

Protein Extraction
Bottom

Proteolysis

Peptide enrichment

MS and MS/MS

Genome annotation
Protein / peptide Up !
identification
 Trypsin

Eksperimen

Precursor masses
Peptide mass fingerprinting

In silico
 5’ 3’ DNA
3’ 5’
Define Set of
Proteins
mRNA
Protein
Select Peptides Proteotypic peptides
MS/MS spectra
Select precursor and Shotgun proteomics LC-MS Targeted MS
product ions 1. Records m/z 1. Select precursor ion
MS MS
Digestion
Validate precursor
and product ions Fractionation
2. Selects peptides based on 2. Specific Precursor fragmentation
abundance and fragments
MS/MS MS/MS
Protein Detection and Homogenization/
Quantitation
3. Protein database search for peptide 3. Use Precursor-Fragment pairs
identification for identification

Data Dependent Acquisition (DDA) Uses predefined set of peptides

Data Independent Acquisition (IDA) for routine typing analyses
 Genome Survey MS Scan
Precursor ions
Experimental

Transcriptome Acquired MS/MS

Product ions
In Silico

Proteome Database survey

MASCOT /
SEQUEST

Species 1 Species 2
Generate proteotypic
species specific mass lists Genome
Precursor ions (speciation)
Common
protein In Silico
Generate MS/MS product sequences
ion species specific mass
lists Species 3 Species 4

MS and MS
Peptide
Analysis
Experimental Fingerprinting
PMF
Peptide biomarkers
based speciation
Preparasi sampel pada Bottom-up proteomics
1. Sampel dicampur air  (1:5) is homogenisasi, sonikasi
2. Protein dalam suspensi diendapkan dengan aseton (1:1)
3. Aseton dipisahkan menghasilkan pellet protein
4. Pelet protein dilarutkan dalam ammonium bicarbonate (pH 8.5).
5. Denaturasi protein pada 120˚C
6. Reduksi protein dengan Dithiothreitol (DTT) dan alkilasi dengan Iodoacetamide IAA
7. Gidesti dengan trypsin pada 40 C selama 24jam. Trypsin memotong ujung C: Lys (K) & Arg (R)

I. II. III. IV.

+
H2O
 QqQ Hybrid Q-Orbitrap
LC-MS LIT or QIT

UltiMate 3000 LC system and Thermo Scientific™

Survey MS Scan
Thermo Scientific™ UltiMate ™ 3000 LC system and Q Exactive™ Hybrid Quadrupole-Orbitrap MS
Thermo Scientific™ TSQ Quantiva MS

Select ions
of interest
(mass range)

Acquired MS/MS
spectra
RT: 0.00 - 90.01
50.29 NL:
100 1.64E10
50.31
50.23 TIC MS
95 50.37 por100msm
s (2)
90
50.17
85

80

75 48.18

70 50.45
51.80
R e l a tiv e A b u n d a n c e

65 48.07
51.84
60
48.04
55
51.91
50

45

40 44.20
14.70
41.13 44.31 51.96
35 14.56 40.82
14.74
40.04 44.41
30 14.84
14.43 14.89 39.94
13.57 44.52 52.02
25 15.03 20.09 20.53 39.78
12.62 15.18 44.63
20 19.75 21.02
23.17 30.20 39.62
12.08 15.57 33.41
29.83 33.83 52.09
15 23.58 44.80
36.83
12.06
52.14
10
52.20
5 52.34
11.86 53.13 67.44 67.71 68.02 76.90 77.16 77.89 84.16 87.97
3.32 5.37 9.05 56.11 61.15
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90
Time (min)

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Discoverer Daemon: Data Processing
Workflows
Annotator in Action
PhosphoRS in Action

• The pRS probability is the likelihood that there is a phosphorylation

• The pRS Site probability is the likelihood of the phosphorylation occurring at each
individual S,T, or Y
Pendekatan Peptide Fingerprinting
Porcine TIC Generate a mass list (MS and MS/MS data)
Sus scrofa

Database search (MASCOT)

MS and MS/MS matching

0 5 10 15 20 25 30 35 40 45
Time (min)
Peptide fingerprinting

Beef Horse
17 specific peptides 23 specific peptides

Using a relatively high 7 429 common

abundance threshold …
unique peptides
identified

Pork Lamb
14 specific peptides 9 specific peptides