This document discusses quantitative mass spectrometry techniques for protein quantitation. It describes both labeling approaches, such as stable isotope labeling with amino acids in cell culture (SILAC) and isobaric tags for relative and absolute quantitation (iTRAQ), as well as label-free approaches including intensity-based measurements, spectral counting, and normalized spectral abundance factor. The labeling approaches involve metabolic, enzymatic, or chemical labeling of proteins prior to mass spectrometry analysis, while label-free methods quantify proteins based on spectral properties without labeling.

1. Protein Quantitation by Mass
Spectrometry
Let’s weigh proteins !
Amit Kumar Yadav
21st Dec 2012

2. Absence of evidence is not
evidence of absence !

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. Relative or Absolute?

5. Quantitative Approaches
Labeling
approaches
Metabolic
(SILAC)
Enzymatic
(O18)
Chemical
(ICAT)
Label-free
approaches
(Unlabeled)
Intensity
based
(MS1)
Spectral
Counting
(MS2)

6. LABELED APPROACHES
Tag and measure !

7. Isotope-Coded Affinity Tag
(ICAT)
http://www.imsb.ethz.ch/researchgroup/rudolfa/research

8. Stable Isotope Labeling with
Amino Acids in Cell
Culture(SILAC)
http://www.biochem.mpg.de/mann/SILAC/index.html

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. 8-plexing with iTRAQ
http://www.sciex.com/products/standards-and-reagents/itraq-
reagents

11. LABEL FREE
Its free !

12. Overview
Less label, more free: approaches in label-free quantitative mass
spectrometry. Proteomics, 11(4), 535–53. doi:10.1002/pmic.201000553

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. 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. RSC
 RSC, = log2 of a ratio of abundance
between two samples

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. 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. 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. 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. Tools for Label free

21. Potential Bias at various steps