![Page 1: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/1.jpg)
Quantitation strategies in mass spectrometry-based proteomics
Fang Liu, Ph.D.March 26, 2020
S U M S FUNDAMENTALS SEMINARS
![Page 2: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/2.jpg)
Submit your questions!
![Page 3: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/3.jpg)
Using proteomics methods to answer:• What are in my samples?
- Qualitative description.- Examples: mysterious gel bands, interacting proteins in IP-MS, etc.
• How much are there in my samples?- Quantitative description.- Examples: biomarker discovery, PTM occupancy rate, etc.
![Page 4: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/4.jpg)
A typical workflow in bottom-up proteomics experiment
Homogenization/ Cell lysis
Proteinprecipitation
Proteolytic digestion
LC-MS/MS
Databasesearch
Cell cultures,Primary cells,
Frozen tissues,FFPE,
Biofluids,Exosomes,
Secretome, etc.
Denature,Reduce,Alkylate.
Raw data
![Page 5: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/5.jpg)
What does LC-MS/MS data look like?
Full scan
Full scan
Full scan
Full scan20 M
S/MS sc
ans
20 MS/M
S scans
20 MS/M
S scans
Full scan
MS/MS scan
LC trace
*
![Page 6: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/6.jpg)
Common quantitation techniques• Label-free
• Spectral counting• Area-based: extracted ion chromatogram (XIC)
• Label-based• Metabolic labeling (e.g. SILAC)• Enzymatic labeling (e.g., trypsin and 18O-labeled water)• Isobaric mass tags (e.g. TMT)• Chemical labeling (e.g. dimethyl labeling, Isotope-Coded Affinity Tag
(ICAT))• Spike-in heavy internal standards (e.g. absolute quantification)
![Page 7: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/7.jpg)
Incorporating quantitation strategies into a typical proteomics workflow
Homogenization/ Cell lysis
Proteinprecipitation
Proteolytic digestion LC-MS/MS
DatabasesearchCell cultures,
Primary cells,Frozen tissues,
FFPE,Biofluids,
Exosomes, Secretome, etc.
Denature,Reduce,Alkylate.
Raw data
MetabolicLabeling
Isobaric taggingChemical labeling (e.g. demethylation)
Internal standards
Label-freeChemical labeling (e.g. ICAT)Internal standards
Enzymatic labeling
![Page 8: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/8.jpg)
Label-free quantitation• Spectral counting• Area-based: extracted ion chromatogram (XIC)
![Page 9: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/9.jpg)
Spectral Counting – Protein abundance estimated by the MS/MS count• Based on the observation that peptides that are more abundant will be
detected and fragmented more often. • Semi-quantitative: Physicochemical properties, protein lengths, peptide
abilities to be ionized may vary.• Fast and cheap: widely used in discovery proteomics (e.g. IP-MS).
Antibody-boundMagnetic beads
YX Y
Incubated cell lysate w/ antibody
Antibody-boundMagnetic beads
Y
Remove unbound proteins
MS analysis
Spec
tral c
ount
Protein X
X Y X Y
Protein Y
Prot
ein
Qua
ntity
X Y
![Page 10: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/10.jpg)
Spectral Counting – Protein abundance estimated by the MS/MS count• Various mathematical normalization strategies exist:
• normalized spectral abundance factor (NSAF). • distributed normalized spectral abundance factor (dNSAF)• exponentially modified protein abundance index (emPAI)• Spectral index (SIN)
McIlwain, Sean, et al., BMC bioinformatics 13.1 (2012): 308.
![Page 11: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/11.jpg)
Example Data – Byonic output
![Page 12: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/12.jpg)
Example Data – Byonic output
![Page 13: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/13.jpg)
Example Data – Byonic output
![Page 14: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/14.jpg)
Example Data – Heatmap
![Page 15: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/15.jpg)
Label-free quantitation• Spectral counting• Area-based: extracted ion chromatogram (XIC)
![Page 16: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/16.jpg)
XIC-based quantitation • Total amount of peptide is determined by the area under curve (AUC)
in the extracted ion chromatogram (XIC).• Variance across multiple LC-MS runs: elution peak alignment, peak
normalization, peak picking, etc.• Often used in targeted methods (e.g. MRM, PRM) for improved
selectivity and sensitivity.
Sample A Sample B A B
![Page 17: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/17.jpg)
XIC-based quantitation used in targeted methodsMultiple Reaction Monitoring (MRM)
Parallel Reaction Monitoring (PRM)
![Page 18: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/18.jpg)
Example Data – Skyline output
![Page 19: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/19.jpg)
Example Data – Skyline output
![Page 20: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/20.jpg)
Label-based quantitation• Metabolic labeling (e.g. SILAC)• Isobaric mass tags (e.g. TMT)
![Page 21: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/21.jpg)
Metabolic Labeling• Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)
• Initially, it allows differential labeling of 2-3 experimental conditions.• 2H4-Lysine, 13C6-Arginine, 15N213C6-Lysine, 15N413C6-Arginine, etc.
Condition 1Lys0, Arg0
Protein Extraction
Denature,Reduce,Alkylate,Digest.
1:1:1Combine
LC-MS/MS
Condition 2Lys4, Arg6
Condition 3Lys8, Arg10
Ong, Shao-En, et al., Molecular & cellular proteomics 1.5 (2002): 376-386.
![Page 22: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/22.jpg)
SILAC• Pros:
• High quantitative accuracy – heavy and light samples combined at early steps, eliminating indeterminate errors during sample handling.
• More total protein material available – good for enrichment techniques (PTM studies, immunoprecipitation, etc.).
• Cons:• Arg to Pro conversion. Solved by controlling heavy Arg concentration
or allowing heavy Pro for quantification.• Heavy isotopes may affect some cell growth and metabolism. • Limited multiplexing capacity. Can be expanded by using one common
“bridge” sample.• Mostly used in cell cultures. Can be expanded to primary cultures and
tissue samples by using SILAC as a ‘spike-in’ internal standard.
Geiger, Tamar, et al., Nature protocols 6.2 (2011): 147.
![Page 23: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/23.jpg)
Spike-in SILAC: Expanding multiplexity and applicability
Individual LC-MS/MS analysis still needed.
Condition 1Light
Protein Extraction
Denature,Reduce,Alkylate,Digest.
1:1:1Combine
LC-MS/MS
Condition 2Light
Condition 3Light
Condition 4Heavy
Ratio of ratio
Krüger, Marcus, et al., Cell 134.2 (2008): 353-364.
m/z
light
heavy
m/z
light heavy
m/z
light heavy
![Page 24: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/24.jpg)
Spike-in SILAC: Expanding multiplexity and applicability
Individual LC-MS/MS analysis still needed.
Condition 1Light
Protein Extraction
Denature,Reduce,Alkylate,Digest.
1:1:1Combine
LC-MS/MS
Condition 2Light
Condition 3Light
Condition 4Heavy
Ratio of ratio
Krüger, Marcus, et al., Cell 134.2 (2008): 353-364.
m/z
light
heavy
m/z
light heavy
m/z
light heavy
![Page 25: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/25.jpg)
Spike-in Standard
Super SILAC
Spike-in SILAC: Expanding multiplexity and applicability
Individual LC-MS/MS analysis still needed.
Condition 1Light
Protein Extraction
Denature,Reduce,Alkylate,Digest.
1:1:1Combine
LC-MS/MS
Condition 2Light
Condition 3Light
Condition 4Heavy
Ratio of ratio
Spike-in Standard
SILAC-mouse *
Krüger, Marcus, et al., Cell 134.2 (2008): 353-364.
m/z
light
heavy
m/z
light heavy
m/z
light heavy
![Page 26: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/26.jpg)
Example Data – MaxQuant output
![Page 27: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/27.jpg)
NeuCode SILAC – Combines SILAC and isobaric tagging• Mass defect: the difference between a compound's exact mass and its
nominal mass resulting from different neutron-binding energies.• Requires specially chosen isotopologues of lysine and high resolving
power.
Hebert, Alexander S., et al., Nature methods 10.4 (2013): 332-334.Overmyer, Katherine A., et al., Nature protocols 13.2 (2018): 293.
![Page 28: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/28.jpg)
NeuCode SILAC – Increased multiplexity for metabolic labeling
• Can also be multiplexed with existing SILAC amino acids.
Hebert, Alexander S., et al., Nature methods 10.4 (2013): 332-334.Overmyer, Katherine A., et al., Nature protocols 13.2 (2018): 293.
![Page 29: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/29.jpg)
Label-based quantitation• Metabolic labeling (e.g. SILAC)• Isobaric mass tags (e.g. TMT)
![Page 30: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/30.jpg)
Isobaric Labeling – Enabling higher sample multiplexing• Isobaric mass tags have identical overall mass but vary in terms of the
distribution of heavy isotopes (13C, 15N) around their structure. • Most common examples: amine-reactive tags
› Tandem Mass Tag (TMT 2plex, 6plex, 10plex, 11plex, 16plex)› Isobaric tags for relative and absolute quantitation (iTRAQ 4plex, 8plex)
https://www.thermofisher.com/
![Page 31: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/31.jpg)
126.127726
127.124761 127.131081
128.128116 128.134436
129.131471 129.137790
130.134825 130.141145
131.138180 131.144499
TMT11plex – Based on mass defect
• Modification mass on MS1 level:229.162932 (monoisotopic)
• Reporter Mass on MS2 level (HCD):(monoisotopic)
• 6.32 mDa mass differences between 15N and 13C isotopes.
![Page 32: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/32.jpg)
MS1 MS2
Example Data – Proteome Discoverer output
![Page 33: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/33.jpg)
Enabling concurrent MS analysis and relative quantification of up to 16 different samples• Samples can be derived from cells, tissues, biological fluids, etc.• Various experimental designs: replicates, time points, etc.
Treatment 1
Treatment 1
Treatment 1
Treatment 2
Treatment 2
Treatment 2
Control
Control
Control
Sample Pool
Protein Extraction
Denature,Reduce,Alkylate,Digest.
Labeling Combine LC-MS/MS
MS1Precursor Ion
SPS-MS3Reporter Ion
5-10 ug total protein for each sample
![Page 34: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/34.jpg)
Enabling concurrent MS analysis and relative quantification of up to 16 different samples• Samples can be derived from cells, tissues, biological fluids, etc.• Various experimental designs: replicates, time points, etc.
Protein Precipitation
Denature,Reduce,Alkylate,Digest.
Labeling Combine LC-MS/MS
MS1Precursor Ion
SPS-MS3Reporter Ion
Savitski, Mikhail M., et al., Science 346.6205 (2014): 1255784.
![Page 35: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/35.jpg)
Isobaric Labeling• Pros:
• Multiplexing capability.• Cons:
• Ratio compression due to isolation interference – improved by SPS-MS3.
Ting, Lily, et al., Nature methods 8.11 (2011): 937.
![Page 36: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/36.jpg)
Protein Quantitation ResultsCluster analysis
Fold changes
Sample v1
Sam
ple
v2
Regression analysisFactor analysis
Motif analysis Network analysis Structural analysis
![Page 37: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/37.jpg)
Thank You
![Page 38: Quantitation strategies in mass spectrometry-based proteomics · Quantitation strategies in mass spectrometry-based proteomics Fang Liu, Ph.D. March 26, 2020 SUMS FUNDAMENTALSSEMINARS](https://reader036.vdocument.in/reader036/viewer/2022062506/5f088ac87e708231d422867d/html5/thumbnails/38.jpg)
More Fundamentals Webinars!• Thursday April 2, 2020
Fundamentals: Measuring concentrations of small molecules using mass spectrometry - theory and practiceSpeaker: Karolina Krasinska, MS – SUMS
• Thursday April 9, 2020Fundamentals: Intact protein mass spectrometry - tips and best practicesSpeaker: Theresa McLaughlin, MS – SUMS
https://mass-spec.stanford.edu/events