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Protein Quantitation by Mass
Spectrometry
Let’s weigh proteins !
Amit Kumar Yadav
21st Dec 2012
Absence of evidence is not
evidence of absence !
Quantitation Bias
Quantitative mass spectrometry in proteomics: a critical review. Analytical
and bioanalytical chemistry,...
Relative or Absolute?
Quantitative Approaches
Labeling
approaches
Metabolic
(SILAC)
Enzymatic
(O18)
Chemical
(ICAT)
Label-free
approaches
(Unlab...
LABELED APPROACHES
Tag and measure !
Isotope-Coded Affinity Tag
(ICAT)
http://www.imsb.ethz.ch/researchgroup/rudolfa/research
Stable Isotope Labeling with
Amino Acids in Cell
Culture(SILAC)
http://www.biochem.mpg.de/mann/SILAC/index.html
Isobaric tags for relative and
absolute quantitation (iTRAQ)
Analysis of protein complexes using mass spectrometry
Nature ...
8-plexing with iTRAQ
http://www.sciex.com/products/standards-and-reagents/itraq-
reagents
LABEL FREE
Its free !
Overview
Less label, more free: approaches in label-free quantitative mass
spectrometry. Proteomics, 11(4), 535–53. doi:10...
Spectral Counting (SpC)
 More abundant peptides will be
selected for fragmentation and will
produce a higher abundance of...
Normalised Spectral Index(SIn)
SIN = a normalised spectral
index
To convert SI into SIn, SI is normalised for
variations b...
RSC
 RSC, = log2 of a ratio of abundance
between two samples
Protein Abundance Index
(PAI)
 An estimate of the protein abundance
in a sample can be calculated using
the protein abund...
Exponentially modified
Protein Abundance Index
(emPAI)
which is directly proportional to the
protein content in a sample
...
Absolute Protein Expression
(APEX)
 Absolute Protein Expression (APEX) is a
modified spectral counting technique that
tak...
Normalised Spectral
Abundance Factor (NSAF)
 Normalised Spectral Abundance Factor
(NSAF) provides an improved measure for...
Tools for Label free
Potential Bias at various steps
proteomics, mass spectrometry, science, bioinformatics, electrophoresis, liquid-chromatography 3.ph d coursework-proteomic...
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proteomics, mass spectrometry, science, bioinformatics, electrophoresis, liquid-chromatography 3.ph d coursework-proteomicsclass-21dec2012

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proteomics, mass spectrometry, science, bioinformatics, electrophoresis, liquid-chromatography 3.ph d coursework-proteomicsclass-21dec2012

  1. 1. Protein Quantitation by Mass Spectrometry Let’s weigh proteins ! Amit Kumar Yadav 21st Dec 2012
  2. 2. Absence of evidence is not evidence of absence !
  3. 3. Quantitation Bias Quantitative mass spectrometry in proteomics: a critical review. Analytical and bioanalytical chemistry, 389(4), 1017–31. doi:10.1007/s00216-007-
  4. 4. Relative or Absolute?
  5. 5. Quantitative Approaches Labeling approaches Metabolic (SILAC) Enzymatic (O18) Chemical (ICAT) Label-free approaches (Unlabeled) Intensity based (MS1) Spectral Counting (MS2)
  6. 6. LABELED APPROACHES Tag and measure !
  7. 7. Isotope-Coded Affinity Tag (ICAT) http://www.imsb.ethz.ch/researchgroup/rudolfa/research
  8. 8. Stable Isotope Labeling with Amino Acids in Cell Culture(SILAC) http://www.biochem.mpg.de/mann/SILAC/index.html
  9. 9. Isobaric tags for relative and absolute quantitation (iTRAQ) Analysis of protein complexes using mass spectrometry Nature Reviews Molecular Cell Biology 8, 645-654 (August 2007)
  10. 10. 8-plexing with iTRAQ http://www.sciex.com/products/standards-and-reagents/itraq- reagents
  11. 11. LABEL FREE Its free !
  12. 12. Overview Less label, more free: approaches in label-free quantitative mass spectrometry. Proteomics, 11(4), 535–53. doi:10.1002/pmic.201000553
  13. 13. Spectral Counting (SpC)  More abundant peptides will be selected for fragmentation and will produce a higher abundance of MS/MS spectra, and is therefore proportional to protein amount in data- dependent acquisition  SpC = the number of MS/MS spectra per peptide
  14. 14. Normalised Spectral Index(SIn) SIN = a normalised spectral index To convert SI into SIn, SI is normalised for variations between protein amounts across data sets by dividing the SI for protein k by the sum of the SI for all proteins in a replicate, and is further normalised by dividing by the length of a protein to account for the expectation that
  15. 15. RSC  RSC, = log2 of a ratio of abundance between two samples
  16. 16. Protein Abundance Index (PAI)  An estimate of the protein abundance in a sample can be calculated using the protein abundance index (PAI), which is defined as the number of observed peptides in the experiment divided by the number of observable tryptic peptides for each protein within a given mass range of the mass spectrometer employed
  17. 17. Exponentially modified Protein Abundance Index (emPAI) which is directly proportional to the protein content in a sample  The protein content can be calculated in terms of a molar percentage by dividing the emPAI value of a protein by the sum of all emPAI values multiplied by 100
  18. 18. Absolute Protein Expression (APEX)  Absolute Protein Expression (APEX) is a modified spectral counting technique that takes into account the number of observed peptide mass spectra for a protein and the probability of the peptides being detected by the MS instrument  The key feature of APEX is Oi, a correction factor for the expectation of observing a tryptic peptide in an experiment, which is calculated by a machine learning classification algorithm based on peptide length and amino acid composition. This technology is based on the findings of Mallick and colleagues in a study involving the prediction of proteotypic peptides
  19. 19. Normalised Spectral Abundance Factor (NSAF)  Normalised Spectral Abundance Factor (NSAF) provides an improved measure for relative abundance by taking into account the length of the protein, which is calculated by dividing the SpC for a protein by its length (L)  This value is then normalised by dividing by the sum of all SpC/L for all proteins in an experiment  The dynamic range for NSAF values is approximately four orders of magnitude, and abundance changes as low as 1.4-fold can be detected
  20. 20. Tools for Label free
  21. 21. Potential Bias at various steps

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