Protein Quantitation by Mass

SpectrometryLet’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, 389(4), 1017–31. doi:10.1007/s00216-007-

1486-6

Relative or Absolute?

Quantitative Approaches

Labeling approaches

Metabolic (SILAC)

Enzymatic (O18)

Chemical (ICAT)

Label-free approaches (Unlabeled)

Intensity based (MS1)

Spectral Counting

(MS2)

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 Reviews Molecular Cell Biology 8, 645-654 (August 2007)

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:10.1002/pmic.201000553

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

Normalised Spectral Index(SIn)

SIN = a normalised spectral

indexTo 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

a longer protein will yield a greater number of

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 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

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

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 Mallickand colleagues in a study involving the prediction of proteotypic peptides

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

Tools for Label free

Potential Bias at various steps