Proteomics Madhura Reka

www.sandorlifesciences.co.
in
PROTEOMICS
AN
OVERVIEW
Madhurarekha Ch. PhD

Head Proteomics & CDFD-SLSPL Grp
madhurarekha@sandor.co.in
Lifesciences Pvt. Ltd.
www.sandorlifesciences.co.in
Black Swallowtail -larvae and butterfly
Same Genome………………Different Proteome

DNA tells what possibly,
RNA what probably and
Proteins what actually happens.

The term proteome and proteomics was coined by Mark Wilkins and colleagues in
the early 1990s.
“Proteomics includes not only the

identification and quantification of
proteins, but also the determination
of their localization, modifications,
interactions, activities, and,
ultimately, their function.”
-Stan Fields in Science, 2001.
“..other than biochemistry done very, very fast, is not entirely clear to me what
proteomics is.” by Marvin Cassman

 Non detection
 Low soluble (basic, hydrophobic,
transmembrane)
 Less abundant
 Range: 2-3 orders of magnitude (pico-
nano) in cell 8 order of magnitude (micro-
femto)
 Identification (sequenced genome)
 Sample complexity (affinity purification )
 Quantification
 Post translational modifications PTMs

PROTEOMICS WORKFLOWS
Gel Based Gel Free

PROTEIN SAMPLE PREPARATION

GEL BASED PROTEOMICS : 2DE & DIGE
Separation  Detection  Diff Quantitation
• cell protein separation and detection

• Staining
• Image Analysis: Quantitative differential
analysis of proteins with high degree of
confidence, sensitivity, linearity
• DIGE: Multiplexing of several samples is
possible without gel to gel variation
• Three fold less gels required to be run

PROTEIN DIGESTION

GEL FREE PROTEOMICS : LCMSMS

MASS SPECTROMETERY
MASS SPECTROMETERS MEASURE MASS/CHARGE RATIOS OF MOLECULES
 Separation
 Ionization
 Isotopes
 Mass spectrum
 Mass Accuracy
 Mass Resolution
 Modifications
 Detection
Sample
+
_
Ionizer Mass Analyzer Detector
John B. Fenn, the originator of electrospray ionization for biomolecules and the 2002 Nobel Laureate in Chemistry

MASS SPECTROMETERY SAMPLES
Macromolecules
Separations
 Identification of proteins, usually in complex mixtures
 Analysis of postranslational and chemical modifications
 Differential expression and quantitation

MASS SPECTROMETERY: MASS ANALYZERS

MASS SPECTROMETERY INSTRUMENT: MALDI TOF TOF

MASS SPECTROMETERY INSTRUMENTS: QTOF

MASS SPECTROMETERY INSTRUMENT: TQD

MASS SPECTROMETERY COMPARISSION

MASS SPECTROMETERY: ION SOURCE
MALDI – Matrix Assisted Laser ESI – Electro Spray

Desorption Ionization Ionization
Ionization
M + H+ = MH+ Protonation : producing a net positive charge of 1+ for every proton added
M-H+ =(M-H)- Deprotonation:net negative charge of 1- is achieved through the removal of a
proton from a molecule
MASS ACCURACY
Mass accuracy in ppm = mass difference * 106

cal. mass
For a compound with a mass of 1000 Daltons:

Eg.
1000 ± 2.0 Da (or ± 2000 ppm) S/TQD
1000 ± 0.5 Da (or ± 500 ppm) TRAP
1000 ± 0.1 Da (or ± 100 ppm) MALDI
1000 ± 0.01 Da (or ± 10 ppm) ESI-TOF
1000 ± 0.002 Da (or ± 2 ppm) FT/ORBI
mass accuracy is the ability to measure or calibrate the instrument

response against a known entity. Usually expressed in parts per million
(ppm), the measurement indicates the deviation of the instrument
response from a known monoisotopic calculated mass.

MASS RESOLUTION
Blue: ∆m = 3.48 R = 1000, 1000 ppm

Red: ∆m = 1.161R = 3000, 333 ppm
Green: ∆m = 0.348R = 10,000, 99 ppm
Black: ∆m = 0.116R = 30,000, 33 ppm
Mass resolution is the dimensionless ratio of the mass of the peak divided by
its width. Usually, the peak width is taken as the full width at half maximum
intensity, (fwhm)

MONOISOTOPIC AND AVERAGE MASS
Peptide Protein
• Monoisotopic value, the mass of the first peak of the isotope distribution
• Average value, equivalent to taking the centroid of the complete isotopic
envelope

INTACT MASS ANALYSIS: MALDI
Data: 1015pl30001.O7 15 Oct 2002 16:22 Cal: 16 Oct 2002 8:12

%Int.
8480.6
100
80
60
5653.4
40
20 16953.0
4239.4
0
5000 10000 15000 20000
Mass/Charge
Sample: 1 pmole apomyoglobin (horse skeletal muscle)

INSTRUMENT: BRUKER ULTRAFLEX III MALDI TOF TOF

INTACT MASS ANALYSIS: LCMSMS
+15
+18+17 +16
100
998.1 1131.0
942.8
1060.4 +14
+19
80 893.2 1211.7
Relative Abundance
+20 +13
848.6
60 1304.8
+21
808.2
+12
40 +22 1413.5
771.5 +11
+23 1541.8 +10 +9
20 +24738.0 1695.9 1884.5
707.4
+25
679.1
0
400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z
ACTUAL SPECTRUM
Sample: 1 pmole apomyoglobin (horse skeletal muscle)
INSTRUMENT: WATERS QTOF SYNAPT G2

INTACT MASS ANALYSIS: ION MOBILTY

INTACT MASS ANALYSIS: DRIFT SCOPE

+1
b = (M+H)1+ - y
Intens. [a.u.] Intens. [a.u.]
0
1
2
3
0
1
2
3
4
5
x10 4
x10 4
174.869 174.862
200
226.955
272.788 272.933
315.901
359.026 359.174
400.073
400
458.132 458.221
515.277 515.253
560.343
R P S V G S
602.326 602.391
600
640.477
711.450
730.130
748.928
798.472
Q L
800
843.480 843.399
881.583
925.497
944.429
960.374
996.711
T S
1000
1031.498 1031.516
1088.608
1102.653
1145.645
1186.949
A H
1200
1239.663 1239.533
1284.606
Y
1345.735
1383.716
1402.755
1400
1432.593
I
1472.283
1515.708 1515.573
L
1585.574
1600
1628.711 1628.728
1681.579
1722.234
1741.752 1742.030
MASS FRAGMENTATION: MS and MSMS
1800
L Sulf
1877.920
STINA_1259_RCAF_1877_DHB_060427\0_E8\0\1877.9200.LIFT\fas t
STINA_1259_1742_DHB_060427\0_B8\0\1742.0300.LIFT\fas t
m /z
y = (M+H)1+ - b +1


‘ Proteomics is extremely valuable for understanding of biological

processes and advancing the field of systems biology.’
2DE-MALDI LC-ESI Different methods develop

different subsets of the
proteome with surprisingly
little overlaps.
Even one and the same
sample analyzed on different
brands of ESI or MALDI
instruments identify
predominantly non-
redundant proteins.
Eventually, analyzing a
statistically sufficient number
of replicates might improve,
but not eliminate this
dilemma.
2DE-ESI LC-MALDI
The perfect workflow is not in sight, yet.




MODIFICATIONS

SOME COMMON POST TRANSLATIONAL MODIFICATIONS

2DE: PHOSPHORYLATION PATTERNS

Silver Staining ImmunoStaining

LC STRATERGIES FOR PHOSPHORYLATED PROTEINS

MALDI TOF/TOF ANALYSIS OF PHOSPHORYLATION
Linear positive peptide Linear negative

peptide
mode mode
phosphopeptide
phosphopeptide

STRATERGIES FOR GLYCOSYLATED PROTEINS

2DE MS LC MS
Maldi TOF/TOF N -Glycosylation Kaji et al., Nat Protoc. 2006;1(6):3019-3027

MALDI TOF/TOF ANALYSIS OF GLYCOSYLATION
Tf glycan Spectrum after PNGase Annotated Spectrum by GlycoQuest (Bruker)

treatment search engine with glycan DB database

MALDI TOF/TOF : BACTERIAL IDENTIFICATION

MALDI TOF/TOF: PHYLOPROTEOMICS
Detection of pap operon expression by

MALDI analysis. E. coli C1a transfected with
either the nonrecombinant (pACYC184) or
the recombinant pap operon vector (pRHU-
845) were analyzed. A spectral peak appears
at 16556.9 Da for E. coli (pRHU- 845); PapA,
the major structural protein of the pap operon
has a calculated mass of 16554 Da based on
the nucleotide sequence.

MALDI TOF/TOF: CLINICAL MAGNETIC BEADS

CLINPROT: preparation,
measurement and visualization
of peptide and protein
biomarkers in context to clinical
proteomics. This system is
comprised of magnetic bead
based sample preparation,
MALDI-TOF mass spectra
acquisition, and a bioinformatics
package for inspection and
comparison of data sets as well
as for the discovery of complex
biomarker pattern models


Protein biomarker discovery and validation: The long and uncertain path to clinical utility. Nat Biotechnol,
24(8): 971-983

Proteomics Madhura Reka

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www.sandorlifesciences.co.

Madhurarekha Ch. PhD

Black Swallowtail -larvae and butterfly

Same Genome………………Different Proteome

Lifesciences Pvt. Ltd.

“Proteomics includes not only the

Lifesciences Pvt. Ltd.

Lifesciences Pvt. Ltd.

Gel Based Gel Free

Lifesciences Pvt. Ltd.

PROTEIN SAMPLE PREPARATION

Lifesciences Pvt. Ltd.

GEL BASED PROTEOMICS : 2DE & DIGE

Separation  Detection  Diff Quantitation

• cell protein separation and detection

Lifesciences Pvt. Ltd.

Lifesciences Pvt. Ltd.

GEL FREE PROTEOMICS : LCMSMS

Lifesciences Pvt. Ltd.

Ionizer Mass Analyzer Detector

Lifesciences Pvt. Ltd.

 Identification of proteins, usually in complex mixtures

 Analysis of postranslational and chemical modifications

 Differential expression and quantitation

Lifesciences Pvt. Ltd.

MASS SPECTROMETERY: MASS ANALYZERS

Lifesciences Pvt. Ltd.

Lifesciences Pvt. Ltd.

Lifesciences Pvt. Ltd.

MASS SPECTROMETERY INSTRUMENT: TQD

Lifesciences Pvt. Ltd.

MASS SPECTROMETERY COMPARISSION

Lifesciences Pvt. Ltd.

MASS SPECTROMETERY: ION SOURCE

MALDI – Matrix Assisted Laser ESI – Electro Spray

Mass accuracy in ppm = mass difference * 106

For a compound with a mass of 1000 Daltons:

mass accuracy is the ability to measure or calibrate the instrument

Lifesciences Pvt. Ltd.

Blue: ∆m = 3.48 R = 1000, 1000 ppm

Lifesciences Pvt. Ltd.

MONOISOTOPIC AND AVERAGE MASS

Lifesciences Pvt. Ltd.

INTACT MASS ANALYSIS: MALDI

Data: 1015pl30001.O7 15 Oct 2002 16:22 Cal: 16 Oct 2002 8:12

Sample: 1 pmole apomyoglobin (horse skeletal muscle)

Lifesciences Pvt. Ltd.

INTACT MASS ANALYSIS: LCMSMS

Sample: 1 pmole apomyoglobin (horse skeletal muscle)

INSTRUMENT: WATERS QTOF SYNAPT G2

Lifesciences Pvt. Ltd.

INTACT MASS ANALYSIS: ION MOBILTY

Lifesciences Pvt. Ltd.

INTACT MASS ANALYSIS: DRIFT SCOPE

Lifesciences Pvt. Ltd.

Lifesciences Pvt. Ltd.

Lifesciences Pvt. Ltd.

‘ Proteomics is extremely valuable for understanding of biological

2DE-MALDI LC-ESI Different methods develop

The perfect workflow is not in sight, yet.

Lifesciences Pvt. Ltd.

Lifesciences Pvt. Ltd.

Lifesciences Pvt. Ltd.

Lifesciences Pvt. Ltd.