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Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Peptide / Protein Quantification
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Outline
• MS Quantification • Why variance matters • Label-free / normalization • Labelled
– iTRAQ
• Peptides to proteins
Terminology
• Absolute Quantification • Relative Quantification • Bias • Variability / Variance • Label-free • Normalization • Labelled • iTRAQ • Isobaric • Reporter ions
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Peptide / Protein Quantification
• Absolute – Estimate the molar amount of protein / peptide in the biological sample – PTMs – Validation
• Relative –Fold change / statistically significant difference between 2 biological states – Biological variation – Biomarker studies
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Label-free
• Area Under Curve – MS1 – Integrate XIC
• Spectral Counting – MS2 – High abundant proteins
Käll L, Vitek O (2011) Computational Mass Spectrometry–Based Proteomics. PLoS Comput Biol 7(12): e1002277. doi:10.1371/journal.pcbi.1002277
MS1 AUC
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
y = f(x)
Area = ∫ 𝑓𝑓(𝑥𝑥)𝑏𝑏𝑎𝑎
XIC for m/z = 421.76
Patient Group A Patient Group B
Prepare Samples
Peptides
Preprocess
Quantify Identify
Analyze Statistically
MS1 & MS2
Spectra
Differentially Abundant
Peptides/Proteins
HPLC-MS/MS
Population Biological
Sample Handling
Instrument
Sources of Bias and Variability
Typical Discovery Experiment
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
MS Quantification
• MS not inherently quantitative • Physiochemical properties invoke different MS
responses • MS only samples a small percentage of total peptides • Bias and variability
Population Biological
Sample Handling
Instrument
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Why Variance Matters
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Normalization
• Remove bias and variability between runs • Global – commonly used
– Median scale – Total ion current (TIC)
• Local – very recent development – Proximity-based intensity normalization (PIN)
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Labeled Quantification
• Run samples simultaneously on in a single run • Add label to samples • Mix samples together • Compute ratios / statistically significant diffs.
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Labeled
• Isobaric – MS2, iTRAQ – Number of samples
• Synthetic Peptides – MS1 – Absolute (AQUA)
• Metabolic – MS1, SILAC – Not higher life forms
Käll L, Vitek O (2011) Computational Mass Spectrometry–Based Proteomics. PLoS Comput Biol 7(12): e1002277. doi:10.1371/journal.pcbi.1002277
www.moffitt.org
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Isobaric Example - iTRAQ
Isobaric Tag Total mass = 305
Reporter Group 113 –119, 121 m/z
Balance Group (?) Mass 184, 186 – 192 m/z
Amine specific peptide reactive group (NHS) N-hydroxysuccinimide O
N
O
ON
N~~~~~~~
N+
N
N+
N
13CH2
15N+
N
13CH2
13CH2
13CH215N+
N
13CH2
13CH2
13CH215N+
15N
13CH3
13CH2
13CH215N+
15N
13CH2
13CH3
13CH2
13CH215N+
N
13CH2
13CH215N+
13CH2
13CH2
15N
13CH2
13CH3
113 114 115 116
117 118 119 121
Applied Biosystems has granted permission to use this slide.
Isob
aric
Rep
orte
r Gro
ups
113
–119
, 121
m/z
iTRAQ® 8-Plex Reagent Chemical Structure
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Reduce, alkylate Cysteines
Trypsin Digest
Reduce, alkylate Cysteines
Trypsin Digest
Reduce, alkylate Cysteines
Trypsin Digest
Reduce, alkylate Cysteines
Trypsin Digest
Proteolytic Digestion
iTRAQ TAG 114
iTRAQ TAG 115
iTRAQ TAG 116
iTRAQ TAG 117
Label peptides with iTRAQ® Reagents
Tissue Images: Rosas HD et al, (2002) Neurology, 58, 695
MIX
2D LC-MS/MS
Normal Brain Tissue-1
Normal Brain Tissue-2
Obtain protein-containing sample, extract protein
Huntington’s Disease-1
Huntington’s Disease-2
iTRAQ Experiment
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900m/z, amu
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
216.2255.1
145.1
428.3
114.1
244.2 782.5513.3
400.2117.1175.1
315.2
598.4499.3185.1212.1
881.6711.4272.2
70.1383.386.1 527.4
199.1 343.2 412.3230.2 326.2 440.3
a1
b1
I
b2
y8
a3
b3
y7
I
a4
b4
y6
I
a5y5
I
y4
y3
y2I
y1
y9(+2)
y9
Inte
nsity
, cou
nts
Peptide = VAIVVGAPRMW = 1024.6249Protein ID = platelet membrane
glycoprotein 11b
“Reporter Ion Mass Tags” from which quantitation is calculated
Peptide iTQVAIVVGAPR MWmono 1024.62 Protein Match Platelet
membrane glycoprotein 11b
Peptide match is made from product ions, e.g., b- and y-ion series
iTRAQ Experiment MS2 Spectrum
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
112.72493 113.91559 115.10626 116.29692 117.48759 118.67826
Mass (m/z)
976.9
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
115.13
114.13 116.13 117.13
113.11No change disease:control
control disease
iTRAQ Results
• Reporter ion intensities reflect relative peptide amounts
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
112.74415 113.88269 115.02123 116.15977 117.29831 118.43685
Mass (m/z)
1.1E +4
0
10
20
30
40
50
60
70
80
90
100
% Int
ensit
y
116.14117.14
115.14
114.14
113.12
112.52129 113.84865 115.17602 116.50338 117.83075 119.15811
Mass (m/z)
3070.1
0
10
20
30
40
50
60
70
80
90
100
% Int
ensit
y
115.12
114.12
116.12117.12
113.11
control
control disease
disease
Increase disease:control
Decrease disease:control
iTRAQ Results • What fold changes are significant?
• Do they represent biological relevance as opposed to
experimental variability?
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
From Käll L, Vitek O (2011) Computational Mass Spectrometry–Based Proteomics. PLoS Comput Biol 7(12): e1002277. doi:10.1371/journal.pcbi.1002277
Labe
led
& L
abel
-free
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
From Peptides to Proteins
Lu, et al., Nature Biotechnology 25, 117 - 124 (2007)
APEX