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Quantitation strategies. Jonathan Trinidad Department of Pharmaceutical Chemistry. Typical Sample Permutations. Gene knockdowns or over expression Inhibitors: eg antibodies or siRNA Growth factors/hormones Cell-cell interactions Drug treatment. Methodology. 2D gel electrophoresis

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Quantitation strategies

Jonathan Trinidad

Department of Pharmaceutical Chemistry


Typical Sample Permutations

Gene knockdowns or over expression

Inhibitors: eg antibodies or siRNA

Growth factors/hormones

Cell-cell interactions

Drug treatment


Methodology

2D gel electrophoresis

silver stain

Fluorescence Difference Gel Electrophoresis

Pro-Q Diamond

2,4-dinitrophenylhydrazine

MS-based quantification methods

“label-free”

stable isotope incorporation

Protein expression array analysis


Quantification using mass spectrometry

Non-isotope methods

Stable isotope methods (MS and MS/MS)

metabolic

enzymatic

chemical

Relative versus absolute quantification

Unstable isotope methods


Factors affecting the accuracy of MS-based quantification

Efficiency/uniformity of labeling

Sample handling variability prior to analysis or sample combination

Resolution in the MS, of the peaks used for quantification

Specificity of given peptides


Label-free estimates of absolute abundance

Many factors influence the detectability of peptides during an LC-MS/MS experiment.

In general, during analysis of complex mixtures, the higher the relative concentration of a given protein, the greater the number of peptides that will be identified from it (and the more intense each of those peptides’ MS intensities).

A number of attempts have been made to roughly estimate a protein’s abundance based upon these parameters.

Rappsilber et al. Genome Res. 2002

Sanders et al. Mol. Cell. Biol. 2002

Ishihama et al. MCP 2005

Silva et al. MCP 2006


How to calculate spectral count?

  • Spectral counting:

    • Spectral abundance factor

    • = (SpC)k/i=1 (SpC)i

N

2. Normalized spectral abundance factor

Old WM, et al Mol Cell Proteomics. 2005 Oct;4(10):1487-502.

Paoletti AC, et al.Proc Natl Acad Sci U S A. 2006 Dec 12;103(50):18928-33.


Label-free relative quantification

Extracted ion chromatography (XIC)-based quantification For each peptide, sum the total signal observed during its elution. Similar to the Beer-Lambert law with 10 caveats. At the protein level, you can add all peptides, or the top three peptides.

Spectra counting

Straightforward in its application. Count each instance of MS/MS acquisition for all the peptides associated with a given protein. Spectra count is “roughly” proportional to relative abundance. Dependent upon IDA-type experiments.


Label-free relative quantification

The accuracy of these approaches is dependent upon several factors:

High mass accuracy is critical for knowing the identify of peptides across runs when MS/MS may not have always been obtained.

The reproducibility of chromatographic analysis is a key parameter.

Chromatographic variations can be addressed after the fact, but XIC quantification is not generally applicable to multi-dimensional analysis.




Proteomic Characterization of the Human Centrosome by Protein Correlation Profiling

  • Discovered several new centrosomal components that may be linked to human disease.

  • Developed a strategy, PCP (protein correlation profiling), that can be used to study other multiprotein complexes.

  • Especially useful for proteins that can’t be purified to homogeneity.

Andersen JS, et al Nature. 2003;426(6966).


Purification and ms analysis procedures
Purification and MS Analysis Procedures Protein Correlation Profiling

Culture human KE37 cells to exponential growth

Isolated centrosomes dissolved in 8M urea buffer

Reduction/Alkylation

Treat with nocodazole and cytochalasin D (arrest in G2/M)

Lys-C/Trypsin Digestion

Hypotonic lysis

Desalted/Concentrated

Sucrose gradient purification and fractionation

Reverse Phase Separation coupled to LC MS/MS

In solution digestion of proteins or 1D SDS-PAGE followed by digestion

Data Analysis using Mascot program (IPI database)

NanoLC MS/MS


1st Validation Method: Immunolocalization

32 of 90 previously uncharacterized proteins identified by MS were tagged with GFP and expressed in U2OS cells.

19 of 32 proteins tested localized to the centrosome



Different experimental designs lead to combination of samples at different points in the analysis


1 samples at different points in the analysisH versus 2H

12C versus 13C

14N versus 15N

16O versus 18O


Metabolic labeling samples at different points in the analysis

Stable Isotope Labeling in Cell Culture (SILAC)


Metabolic labeling samples at different points in the analysis

15N labeling can be used, or alternatively specific amino acids (generally arginine or lysine)

Ideally, the cells will be grown for a number of generations to insure complete incorporation of the isotopic amino acid(s).

yeast, e. coli; mammalian cell lines;

C. elegans; D. melanogaster; rattus rattus

(Krijgsveld 2003, Wu et al. 2004)


Preparation For SILAC Experiments samples at different points in the analysis

Ong SE, Mann M. Nat Protoc. 2006;1(6):2650-60.


Quantitation of Protein Ratios from Peptide Doublets samples at different points in the analysis

Blagoev B et al. Nat Biotechnol.


Strategy to Study Activated EGFR Complex Using SILAC samples at different points in the analysis

Blagoev B et al. Nat Biotechnol.


Multiple SILAC experiments can be combined to create timecourses

Blagov et al.

Nat Bio 2004


Temporal Changes in the Nucleolar Proteome Upon Transcriptional Inhibition

Andersen JS, et al, Nature. 2005


Quantification of the synaptosomal proteome of the rat cerebellum during post-natal development

Feed mice a diet consisting entirely of 15N as the only nitrogen source

McClatchy et al Genome Research 2007


SILAC Mouse for Quantitative Proteomics Uncovers Kindlin-3 as an Essential Factor for Red Blood Cell Function

Kruger et al Cell 2008


Top-Down Quantitation and Characterization of SILAC-Labeled Proteins

13C615N4-Arg

13C614N2-Lys

Waanders, L.F. et al. J Am Soc Mass Spectrom. 2007


Enzymatic labeling Proteins

Carboxypeptidases (e.g. trypsin) can incorporate two oxygen molecules

Aminopeptidases (e.g. Lys-N) can incorporate one oxygen molecule

Enzymatic digests are self limiting


Enzymatic labeling Proteins

The carbonyl oxygen exchange reaction has proven difficult to optimize, resulting in peptides with variable levels of incorporation. This complicates quantitation.




ICAT Proteins

Isotope coded affinity tag

Gygi et al 1999


Cleavable ICAT Proteins

Acid cleavable linker facilitates release

Heavy and light carbon allows for co-eluting peptides

So user-friendly, knowledge of the structure is not required


iTRAQ Proteins

Isobaric Tags for Relative and Absolute Quantitation

Ross et al MCP 2004



Zoom in view of the iTRAQ ion region spectra

showing approximately 6-fold more signal in the peptide from m/z 117 versus m/z 116



A schematic of the new 8-channel (8-plex) iTRAQ reagent spectra

Pierce A, et al. Mol Cell Proteomics. 2008




Absolute quantification approach

Absolute quantification is relative quantification using synthetic isotopes of known concentration.

These can be synthesized in a traditional fashion (AQUA). Purification and quantification of the standards is often cost prohibitive.

Recently, quantified microsynthesized isotopic standards have become commercially available.

For unmodified peptides, QconCAT can be used to synthesize large numbers of isotopic peptides.


Absolute Quantitation Using Synthetic Proteins approach

- QconCAT or Peptide-concatenated standard (PCS)

Pratt JM, et al. Nat Protoc. 2006;1(2):1029-43. ;

Kito K, et al. J Proteome Res. 2007 Feb;6(2):792-800

Kito K, et al. J Proteome Res. 2007 Feb;6(2):792-800


Misc. approach


Differential enrichment using an array of drug-coated beads can be used to identify target protein complexes.

Oda et al.

Anal. Chem. 2003


Changes in the composition of macromolecular complexes can be examined as a function of molecular state

Ranish et al.

Nat Gen 2003


Turnover rates for individual proteins can be determined using isotopic labeling

Pratt et al.

MCP 2002


Retrospective Birth Dating of Cells in Humans using isotopic labeling

Using bomb pulse 14C levels and accelerator mass spectrometry to calculate the age of cells in the body

Spalding et al Cell 2005


Selective reaction monitoring using isotopic labeling

Selective reaction monitoring is conducted on a triple-quadrupole mass spectrometer. It requires a list of known targets, the m/z values of the precursor mass and the m/z of prominent fragment ions. These can be empirically determined or theoretically generated using algorithms.

Q1 is set to selectively pass a specific precursor m/z.

Q2 is set as the collision cell.

Q3 is set to selectively pass a specific fragment ion.

The scans can be quick, on the order of 5 msec. 400 different SRMs can therefore be analyzed every 2 seconds. If the LC retention time is know, these could be scheduled to allow for the acquisition of several thousand SRMs in a single run. This technique can be used in a label-free fashion, used with SILAC or isotopic standards.


Extracting biological insight from quantitative protein lists is the difficult part. A number of approaches have been developed, and have initially been applied to microarray data.


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