1. 1 I. Introduction 1.Definition: Protein Characterization/Proteomics i.Classical Proteomics ii.Functional Proteomics 2.Mass spectrometery I.Advantages in Studying Proteins II.General configuration

2. 2 I. Introduction Why is proteomics necessary? ( Pandey, A., Mann, M., Nature, 405, 837-846, 15June2000 ) complete sequences of genomes is not sufficient to elucidate biological function existence of an open reading frame in genomic data doesn’t imply the existence of a functional gene (8% error in annotations for 340 genes from theMycoplasma genitalium genome) verification of gene product is an important first step in genome annotating modifications of proteins are not apparent from the DNA sequence (isoforms, post-translational modifications) mRNA may or may not correlate with protein level localization of gene product can be determined protein-protein interaction and molecular composition of cellular structures such as organelles can be determined only at the protein level

3. 3 I.1.Definition: Protein Characterization/Proteomics “Proteome” (Wilkins and Williams) entire protein complement of a given genome “Proteomics” naturally: study of the proteome catalog and characterize these proteins large scale analysis of proteins within a single experiment (or series thereof) Proteomics is classified into two disciplines  classical proteomics  functional proteomics Protein Characterization one protein at a time

4. 4 Definition: Classical Proteomics proteome of two (or more) differentially treated cell (tissue) lines are separated/visualized by 2D gel electrophoresis proteins that differ in abundance between the gels are identified by mass spectrometry I.1.iClassical Proteomics

5. 5 Advantages requires little/no knowledge about the proteome is conceptually simple Drawbacks 2D gels usually only allow visualization of ca. 20% of loaded proteins only proteins of MW 10-100 kDa migrate well in 2D gels membrane proteins are not separated well by 2D PAGE determine changes in protein pattern is different because of the inherent high variability of 2D gels proteins in low quantities are not detectable on 2D gels. Increasing the loading material causes lost of separating power

6. 6 I.1.iiFunctional Proteomics Definition: Functional Proteomics subset of proteins is being isolated from a given starting material and identified by mass spectrometry proteins have a common feature which is used for separation (affinity chromatography) common feature combined with bioinformatics can give evidence of the function of the identified proteins

7. 7 I.1.iiFunctional Proteomics Advantages knowledge of protein-protein interactions helps place novel proteins in their functional context (biochemical pathway, components of multi-protein complex) differences are much easier to detect in differential experiments because of reduced subset of proteins sensitivity in terms of detection/identification is high due to the enrichment by affinity purification Drawbacks requires an assumption about a given system requires skills over a very broad range of disciplines

8. 8 I.2.Mass Spectrometry Definition/Requirement: Mass Spectrometry technique to determine the relative weight of atoms and molecules by separation of charged atoms and molecules based (ions) on their mass in the gas phase. (first mass spectrometer 1910, Ne-isotope 20/22) molecules need to be in the vapor phase molecules need to be ionized

9. 9 I.2.i.Advantages in Studying Proteins  High mass accuracy 10 ppm: 1000 Da ± 0.01 Da (UNC)  Identification of proteins via database searching  Detection of post-translational modifications  High sensitivity femto-mol (=50 pg of 50 kDa protein) (UNC)  Study proteins at physiological level  Provides sequence information  Identification of modification sites

10. 10 I.2.ii.General configuration ion source: ionization and transfer of molecules into the gas phase mass analyzer:separation of the molecules due totheir mass ion source mass analyzerdetector

11. 11 II. Mass Spectrometry 1.Analytical Parameters/Definitions i.Molecular weight ii.Mass Accuracy iii.Chemical Background vs. Peak iv.Mass Resolution

12. 12 II.1.i.Molecular weight Mono-isotopic molecular weight: mass of the molecule which elementary composition possesses only the most natural abundant isotopes ( 12 C, 1 H, 16 O, 14 N, etc.) Average-isotopic molecular weight: calculated mass of the molecule out of a elementary composition possesses isotopes in the proportion corresponding to their natural abundances

13. 13 II.1.i.Molecular weight Masses and Abundance of isotopes of natural elements: ElementO#M#MassRelative abundance Average mass C61212.00000098.9012.011 1313.0033551.10 H111.00782599.9851.00794 22.0141020.015 O81615.99491599.76215.9994 1716.9991310.038 1817.9991590.200 N71414.00307499.63414.0067 1515.0001090.366 S163231.97207295.0232.066 3332.9714590.75 3433.9678684.21 3635.9670790.02

14. 14 II.1.i.Molecular weight Expected mass: Acetic acid: C 2 H 4 O 2 Isotopes: 12 C, 13 C, 1 H, 2 H, 16 O, 17 O, 18 O Monoisotopic: 12 C 2 1 H 4 16 O 2 90 possible formulas 6 formulas with significant abundances compositionmassrelative abundance 12 C 2 1 H 4 16 O 2 60.02113100.000 12 C 13 C 1 H 4 16 O 2 61.024482.224 12 C 2 1 H 4 16 O 17 O61.025340.076 12 C 2 1 H 3 2 H 16 O 2 61.027410.060 12 C 2 1 H 4 16 O 18 O62.025380.401 13 C 2 1 H 4 16 O 2 62.027840.012

15. 15 II.1.ii.Chemical Background vs. Peak Definition: peak: must be at least twice the baseline; S/N > 2 peak: more than one data point is needed to define a peak chemical background: chemical must be evaluated to show peak comes from sample

16. 16 II.1.ii.Chemical Background vs. Peak Molecular weight peak width increases with mass: PeptideMass MH + Rel. abundance Leu 5 -enkephalin C 28 H 38 N 5 O 7 556.28 557.28 558.28 559.29 100.00 34.04 6.89 0.89 monoisotopic 556.64 average mass PNGF fragment C 89 H 140 N 27 O 26 2003.05 2004.05 2005.05 2006.05 2007.05 2008.06 88.61 100.00 60.14 25.26 7.78 1.68 monoisotopic base peak 2004.26 average mass Ubiquitin C 378 H 630 N 105 O 118 S 8560.62 8561.63 8562.63 8563.63 8564.63 8565.64 8566.64 8567.64 8568.65 8569.65 8570.65 8571.66 4.14 19.02 47.27 78.47 98.75 100.00 86.58 63.62 40.39 22.09 8.81 3.19 monoisotopic base peak 8565.89 average mass

17. 17 II.1.iii.Mass Accuracy Mass accuracy mass accuracy = ΔM (calculated-observed) in Da, amu, ppm (parts per million) ppm: [(m/z obs -m/z calc )/m/z calc ] x 10 6 Mass spectrometerMass accuracy for peptides FTICRhighest~ 1-5 ppm TOFhigh~ 10-50 ppm Ion-trap/quadrupolesmoderate/low> 500 ppm

18. 18 II.1.iv.Mass Resolution Definition: Resolution = R = M/ΔM ΔM : width at half height estimating unit resolution:M/0.5 estimating complete isotopic (M, M’) resolution: ([M+M’]/2)/([M-M’] x 0.5) ΔM m/z

19. 19 II.1.iv.Mass Resolution Example acetic acid (see slide 14): “unit” resolution: 60 Da and 61 Da  R=60/0.5=120 “complete” isotopic resolution: 61.02448 Da and 61.02534 Da  R=61.02491/0.00043=141,918

20. 20 II.1.iv.Mass Resolution Resolution illustrated: angiotensin II C 50 H 73 N 13 O 12 (from K.G. Owens, M.M. Vestling “Fundamentals and Applications of MALDI-TOF-MS”, A Short Course Spnosored by the American Society for Mass Spectrometry)

21. 21 II.1.iv.Mass Resolution Resolution illustrated: ubiquitin C 378 H 630 N 105 O 118 S (from K.G. Owens, M.M. Vestling “Fundamentals and Applications of MALDI-TOF-MS”, A Short Course Spnosored by the American Society for Mass Spectrometry)