1. Quantitation strategies
Jonathan Trinidad
Department of Pharmaceutical Chemistry

2. Typical Sample Permutations
Gene knockdowns or over expression
Inhibitors: eg antibodies or siRNA
Growth factors/hormones
Cell-cell interactions
Drug treatment

3. 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

4. Quantification using mass spectrometry
Non-isotope methods
Stable isotope methods (MS and MS/MS)
metabolic
enzymatic
chemical
Relative versus absolute quantification
Unstable isotope methods

5. 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

6. 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
Ishihama et al. MCP 2005
Silva et al. MCP 2006

7. 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 Oct;4(10):
Paoletti AC, et al.Proc Natl Acad Sci U S A Dec 12;103(50):

8. 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.

9. 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.

10. Peaks in replicate LC-MS runs can be aligned using software algorithms
Prakash et al.
MCP 2006

11. Peaks in replicate LC-MS runs can be aligned using software algorithms
Prakash et al.
MCP 2006

12. 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).

13. Purification and MS Analysis Procedures
Culture human KE37 cells to exponential growth
Isolated centrosomes dissolved in 8M urea buffer
Treat with nocodazole and cytochalasin D (arrest in G2/M)
Reduction/Alkylation
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

14. 1st Validation Method: Immunolocalization
500 proteins identified in peak centrosome fraction (7).
47 out of 60 known centrosomal proteins identified
90 uncharacterized proteins identified
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

15. Protein correlation profiling of the human centrosome

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

17. 1H versus 2H
12C versus 13C
14N versus 15N
16O versus 18O

18. Stable Isotope Labeling in Cell Culture (SILAC)
Metabolic labeling
Stable Isotope Labeling in Cell Culture (SILAC)

19. Metabolic labeling
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)

20. Preparation For SILAC Experiments
Ong SE, Mann M. Nat Protoc. 2006;1(6):

21. Quantitation of Protein Ratios from Peptide Doublets
Blagoev B et al. Nat Biotechnol.

22. Strategy to Study Activated EGFR Complex Using SILAC
Blagoev B et al. Nat Biotechnol.

23. Multiple SILAC experiments can be combined to create timecourses
Blagov et al.
Nat Bio 2004

24. Temporal Changes in the Nucleolar Proteome Upon Transcriptional Inhibition
Andersen JS, et al, Nature. 2005

25. 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

26. SILAC Mouse for Quantitative Proteomics Uncovers Kindlin-3 as an Essential Factor for Red Blood Cell Function
Kruger et al Cell 2008

27. Top-Down Quantitation and Characterization of SILAC-Labeled Proteins
13C615N4-Arg
13C614N2-Lys
Waanders, L.F. et al. J Am Soc Mass Spectrom. 2007

28. Enzymatic labeling
Carboxypeptidases (e.g. trypsin) can incorporate two oxygen molecules
Aminopeptidases (e.g. Lys-N) can incorporate one oxygen molecule
Enzymatic digests are self limiting

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

30. Many flavors of chemical labeling

31. Some isotopic labeling reagents

32. Isotope coded affinity tag
ICAT
Isotope coded affinity tag
Gygi et al 1999

33. Cleavable ICAT Acid cleavable linker facilitates release
Heavy and light carbon allows for co-eluting peptides
So user-friendly, knowledge of the structure is not required

34. Isobaric Tags for Relative and Absolute Quantitation
iTRAQ
Isobaric Tags for Relative and Absolute Quantitation
Ross et al MCP 2004

35. iTRAQ quantification information is contained in the MS/MS spectra

36. 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

37. iTRAQ can be used to construct four datapoint timecourses
Zhang et al.
MCP 2005

38. A schematic of the new 8-channel (8-plex) iTRAQ reagent
Pierce A, et al. Mol Cell Proteomics. 2008

39. An example of Protein Quantitation Using 8-plex iTRAQ Reagents

40. Tandem mass tags present an alternative multiplexing approach
Thermo

41. 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.

42. Absolute Quantitation Using Synthetic Proteins
- QconCAT or Peptide-concatenated standard (PCS)
Pratt JM, et al. Nat Protoc. 2006;1(2): ;
Kito K, et al. J Proteome Res Feb;6(2):
Kito K, et al. J Proteome Res Feb;6(2):

43. Misc.

44. Differential enrichment using an array of drug-coated beads can be used to identify target protein complexes.
Oda et al.
Anal. Chem. 2003

45. Changes in the composition of macromolecular complexes can be examined as a function of molecular state
Ranish et al.
Nat Gen 2003

46. Turnover rates for individual proteins can be determined using isotopic labeling
Pratt et al.
MCP 2002

47. Retrospective Birth Dating of Cells in Humans
Using bomb pulse 14C levels and accelerator mass spectrometry to calculate the age of cells in the body
Spalding et al Cell 2005

48. 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 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.

49. 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.