Quantitation strategies

Jonathan TrinidadDepartment 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 electrophoresissilver stainFluorescence Difference Gel ElectrophoresisPro-Q Diamond2,4-dinitrophenylhydrazine

MS-based quantification methods“label-free”stable isotope incorporation

Protein expression array analysis

Non-isotope methods

Stable isotope methods (MS and MS/MS)

metabolicenzymaticchemical

Relative versus absolute quantification

Unstable isotope methods

Quantification using mass spectrometry

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

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. 2002Sanders et al. Mol. Cell. Biol. 2002Ishihama et al. MCP 2005Silva et al. MCP 2006

Label-free estimates of absolute abundance

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

1. Spectral counting:

Spectral abundance factor= (SpC)k/i=1 (SpC)i

N

2. Normalized spectral abundance factor

How to calculate spectral count?

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

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.

Label-free relative quantification

Prakash et al.MCP 2006

Peaks in replicate LC-MS runs can be aligned using software algorithms

Prakash et al.MCP 2006

Peaks in replicate LC-MS runs can be aligned using software algorithms

• 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.

Proteomic Characterization of the Human Centrosome by

Protein Correlation Profiling

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

Purification and MS Analysis Procedures

Culture human KE37 cells to exponential growth

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

Hypotonic lysis

Sucrose gradient purification and fractionationIn solution digestion of proteins or 1D SDS-PAGE followed by digestionNanoLC MS/MS

Isolated centrosomes dissolved in 8M urea buffer

Reduction/Alkylation

Lys-C/Trypsin Digestion

Desalted/Concentrated

Reverse Phase Separation coupled to LC MS/MS

Data Analysis using Mascot program (IPI database)

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

1st Validation Method: Immunolocalization

• 500 proteins identified in peak centrosome fraction (7).

• 47 out of 60 known centrosomal proteins identified

• 90 uncharacterized proteins identified

Protein correlation profiling of the human centrosome

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

1H versus 2H12C versus 13C14N versus 15N16O versus 18O

Metabolic labeling

Stable Isotope Labeling in Cell Culture (SILAC)

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)

Metabolic labeling

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

Preparation For SILAC Experiments

Quantitation of Protein Ratios from Peptide Doublets

Blagoev B et al. Nat Biotechnol.

Strategy to Study Activated EGFR Complex Using SILAC

Blagoev B et al. Nat Biotechnol.

Blagov et al. Nat Bio 2004

Multiple SILAC experiments can be combined to create timecourses

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

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

13C615N4-Arg

13C614N2-Lys

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

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

Enzymatic labeling

Enzymatic digests are self limiting

Enzymatic labeling

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

Many flavors of chemical labeling

Some isotopic labeling reagents

ICATIsotope coded affinity tag

Gygi et al 1999

Cleavable ICAT

Acid cleavable linker facilitates releaseHeavy and light carbon allows for co-eluting peptidesSo user-friendly, knowledge of the structure is not required

iTRAQIsobaric Tags for Relative and Absolute Quantitation

Ross et al MCP 2004

iTRAQ quantification information is contained in the MS/MS spectra

Zoom in view of the iTRAQ ion region showing approximately 6-fold more signal in

the peptide from m/z 117 versus m/z 116

Zhang et al.MCP 2005

iTRAQ can be used to construct four datapoint timecourses

Pierce A, et al. Mol Cell Proteomics. 2008

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

An example of Protein Quantitation Using 8-plex iTRAQ Reagents

Tandem mass tags present an alternative multiplexing approach

Thermo

Absolute quantification

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- QconCAT or Peptide-concatenated standard (PCS)

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

Pratt JM, et al. Nat Protoc. 2006;1(2):1029-43. ; Kito K, et al. J Proteome Res. 2007 Feb;6(2):792-800

Misc.

Oda et al.Anal. Chem. 2003

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

Ranish et al.Nat Gen 2003

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

Pratt et al.MCP 2002

Turnover rates for individual proteins can be determined using isotopic labeling

Retrospective Birth Dating of Cells in Humans

Spalding et al Cell 2005

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

Selective reaction monitoring

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.