1. Mass spectrometry-based proteomics Jeff Johnson Feb 19, 2014

2. MS Proteomics in a Nutshell • Ionization • Delivering macromolecules to the MS • Ion Manipulation • Trapping and ejecting analytes of interest • Fragmentation • Breaking apart for more information • Mass analysis and detection • Measuring masses and quantifying intensities

3. MS Proteomics in a Nutshell • Ionization • Delivering macromolecules to the MS • Ion Manipulation • Trapping and ejecting analytes of interest • Fragmentation • Breaking apart for more information • Mass analysis and detection • Measuring masses and quantifying intensities

4. Macromolecular Ionization for MS • Analyte must be in the gas phase for mass analysis • Analyte must be charged in order to be manipulated by electric and magnetic fields • All mass analyzers measure mass-to-charge ratios (m/z) • Two predominant approaches (shared the Nobel prize in 2002) • Matrix assisted laser desorption ionization • Electrospray ionization

5. MALDI Ionization • Sample is spotted in a matrix that readily absorbs UV/IR light and is vaporized by a laser • Common matrix: 2,5-dihydroxybenzoic acid (DHB) • Advantages • Fast and easy • Spots can be reanalyzed later • Most analytes get one +ive charge makes it easy to deconvolute • Disadvantages • Harsh. Often breaks analytes apart (e.g., breaks phosphorylation) • Not easily combined with online HPLC separations

6. Electrospray Ionization • Sample is dissolved in liquid and pushed through a charged needle and sprayed into an evaporation chamber • Droplets pulled into the MS source by electric potential between the needle and the MS • Heated ion transfer tube evaporates water molecules in droplets leaving +ively charged analytes in the gas phase • Advantages • Compatible with online HPLC separations • “Soft” ionization maintains label and non-covalent interactions • Disadvantages • Analytes can have different numbers of charges, can be difficult to deconvolute without high mass accuracy • Different samples going through the same electrospray tip causes carryover problems • Especially bad with online HPLCs

7. Ionization is Nearly Impossible to Predict • Different molecules ionize with different efficiencies and are very difficult to predict • MS intensity ratios between different molecules do not reflect ratios in the sample from which they were derived • Most quantification by MS is relative 2x B X A B A

8. Ionization is Nearly Impossible to Predict Sample 1 Sample 2 • Different molecules ionize with different efficiencies and are very difficult to predict • MS intensity ratios between different molecules do not reflect ratios in the sample from which they were derived • Most quantification by MS is relative A A Sample 1 Sample 2 A A * Assumption: MS run 1 = MS run 2

9. MS Proteomics in a Nutshell • Ionization • Delivering macromolecules to the MS • Ion Manipulation • Trapping and ejecting analytes of interest • Fragmentation • Breaking apart for more information • Mass analysis and detection • Measuring masses and quantifying intensities

10. Ion Manipulation • We need a way to select only ions of interest • Most detectors are just electron multipliers that don’t measure mass but just detect a thing hitting the multiplier • We can manipulate ions to deliver defined mass ranges to the detector to get a mass spectrum • Two common tools: • Ion traps • Quadrupoles • Both use electric and magnetic fields to select ions of a particular m/z range

11. Ion Trap • Ions are trapped by 3D electric field by DC and AC applied to the electrodes • An ion trap can accumulate ions as they come in from the source and store them • Low resolution: +/- 1 Da

12. Quadrupole • Can be thought as a mass filter • DC and AC fields applied that stabilize a trajectory for ions in a desired mass range, undesired ions are ejected • Quadrupole operate with a continuous flow of ions • Low resolution (+/- 1 Da)

13. MS Proteomics in a Nutshell • Ionization • Delivering macromolecules to the MS • Ion Manipulation • Trapping and ejecting analytes of interest • Fragmentation • Breaking apart for more information • Mass analysis and detection • Measuring masses and quantifying intensities

14. Fragmentation • Usually measuring the mass of an analyte is not enough to conclusively identify it • By fragmenting an analyte and measuring the masses of the fragments we can obtain further information to identify the analyte • There are many types of fragmentation but collision-induced dissociation (CID) is the most common • Fastest and most generally successful for the widest variety of proteins and peptides

15. Collision-Induced Dissociation • Give ions kinetic energy and collide with gas molecules (He) • Collisions build up potential energy until a fragmentation event can occur • Ideally potential energy is strong enough to break a single peptide bond but not strong enough to fragment further • Can be done in an ion trap or a quadrupole

16. Collision Induced Dissociation A E P T I R H2O Fragment (somewhat) randomly along the peptide backbone

17. B-type Ions A E P T A E P A E Intensity 399.2 A 298.1 201.1 72.0 M/z

18. Y-type Ions R I T P E A H2O Intensity M/z

19. B-type,A-type,Y-type Ions R I T P E A H2O Intensity M/z

20. MS Proteomics in a Nutshell • Ionization • Delivering macromolecules to the MS • Ion Manipulation • Trapping and ejecting analytes of interest • Fragmentation • Breaking apart for more information • Mass analysis and detection • Measuring masses and quantifying intensities

21. Mass Analysis and Detection • All mass analyzers achieve the same thing: physical separation based on mass:charge • Magnetic sector is the simplest and one of the earliest types Magnetic Sector MS

22. FT-ICR MS • FT-ICR = Fourier transform – ion cyclotron resonance • Ion injected in line with a strong magnetic field that induces a cyclical motion • Radius of the cyclotron motion is proportional to m/z

23. Time-of-flight MS Medium / High Resolution

24. Quadrupoleand Ion Trap MS Electron multiplier • You can use a quadrupoles or ion traps to “scan out” ions across an entire mass range to a detector by gradually ramping voltages • Low resolution but electron multipliers make these very sensitive

25. Orbitrap MS • Characteristic frequencies: • Frequency of rotation ωφ • Frequency of radial oscillations ωr • Frequency of axial oscillations ωz r z φ

26. Power of Fourier Transforms • FTs convert from time domain to freq domain • Instead of a single measurement the m/z is measured over a period of time and the FT essentially averages all those measurements • Resulting data is very high resolution

28. Chromatography to Simplify Complexity Complex Sample MS • Complexity hurts sensitivity • A constant, defined number of ions can be analyzed in each MS scan • Sensitivity is constant (around 1 fmol) • A scan with fewer ions is more sensitive than a scan with many

29. Chromatography to Simplify Complexity MS HPLC Complex Sample C18 RP column ACN gradient A C B A B D C D

30. Chromatography to Simplify Complexity Very Complex Sample Offline HPLC (e.g., SCX) SCX Fractions SCX Fractions Injected individually MS Online HPLC (RP)

31. Acquiring MS Data • Data acquisition depends on experimental goals • Data-dependent acquisition • MS attempts to acquire data to allow you to identify a maximum number of unknowns • Commonly used for analyses where you don’t know what you’re looking for • Targeted acquisition • MS only acquires data for what you tell it to acquire • Much more sensitive than data-dependent, but also more limited in scope

32. Data-Dependent Acquisition

33. Data-Dependent Acquisition 1 2 3 High resolution survey scan (<5 ppm mass accuracy)

34. Data-Dependent Acquisition Low resolution MS/MS scan 1

35. Data-Dependent Acquisition Low resolution MS/MS scan 2

36. Data-Dependent Acquisition Low resolution MS/MS scan 3

37. Peptide Identification AA sequence DB (Species UniProt)

38. Peptide Identification AA sequence DB (Species UniProt) 1 AA DBs restricted by parent ion massmeasured in survey scan 2 3

39. Peptide Identification AA sequence DB (Species UniProt) MS/MS 1 1 AA DBs restricted by parent ion massmeasured in survey scan 2 MS/MS 2 3 MS/MS 3

40. Probabilistic Matching (X!Tandem) # of Matches Second Best Best Hit by-Score= Sum of intensities of peaks matching B-type or Y-type ions HyperScore= Hyper Score

41. Model spectrum comparisons

42. Pattern Matching (Sequest)

43. SequestXCorr Cross Correlation (direct comparison) Auto Correlation (background) Correlation Score Offset (AMU) XCorr =

44. Targeted Acquisition with a QQQ SRM Assay • A priori knowledge required: • SRM assay development for a list of proteins/peptides of interest • Information derived from label-free unbiased proteomic analysis

45. “Sensitivity” • Sensitivity of a MS is well defined, but the ability to identify something is a very different concept • Ability to detect depends on: • Sample complexity • MS sensitivity • MS speed • A faster MS can collect go deeper in each survey scan • Think “top 10” vs. “top 50” • MS mass accuracy • Better mass accuracy improves the ability to identify peptides but sacrifices speed and MS sensitivity • Especially important for variable modifications • The “best” method is very dependent on the experimental goals

46. Database Searching Ion trap +/- 1 Da Orbitrap +/- 0.002 Da Database “search space”

47. Database Searching Ion trap +/- 1 Da Orbitrap +/- 0.002 Da Database “search space” +S/T/Y phosphorylation

48. Database Searching Ion trap +/- 1 Da Orbitrap +/- 0.002 Da Database “search space” +S/T/Y phosphorylation

49. Protein Quantification • A mass spectrometer is an inherently quantitative device but the ionization source is not • Different peptides/proteins are ionized with drastically different efficiencies • Absolute abundances in a mass spectrometer are not precisely indicative of abundance in a sample • Solution: stable isotope labeling • Compare samples that have been labeled with stable isotopes (13C, 15N, 2H) • ‘Heavy’ isotopes behave chemically identically to their ‘light’ counterparts but are separated in the MS

50. Isotope Coded Affinity Tag (ICAT)