Protein Identification Techniques Summary Layout. 4 Edamn sequencing Peptide mass fingerprinting & peptide sequencing. Q-Tof Micro

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1 5/18/2014 Application of mass spectrometry in proteomic science Neda Saraygord-Afshari; PhD 1 2 Protein Identification Techniques Summary Layout Different methods are used for protein sequencing/identification. As the most famous: On overview of protein identification techniques 1 Sanger sequencing A brief history of MS development 2 Maxim-Gilbert sequencing How MS instruments work 3 MALDI TOF/TOF MS 4 Edamn sequencing Peptide mass fingerprinting & peptide sequencing 5 6 Top-down vs. bottom-up proteomics 3 Mass Spectrometry (MS) 4 Q-Tof Micro MALDI-TOF The history of mass spectrometry 1906 Wilhelm Wien 1898The foundation of MS systems Thomson is awarded the Nobel Prize in Physics "in recognition of the great merits of his theoretical and experimental investigations on the conduction of electricity by gases 1922 Aston is awarded the Nobel Prize in chemistry "for his ESI-TOF discovery, by means of his mass spectrograph, of isotopes, in a large number of non-radioactive 1988 MALDI revolutionized biological MS Thomson 1913 achieved successful separation of Ne isotopes Tandem mass spectrometer appears Wolfgang Paul 1953 invention of quadrupole and iontrap elements, and for his enunciation of the wholenumber rule Lawrence receives the Nobel Prize in Physics for the cyclotron FT-ICR LTQ-Orbitrap Wolfgang Paul receives the Nobel Prize in Physics "for the development of the ion trap technique" John Bennett Fenn and Koichi Tanaka are awarded one-quarter of the Nobel Prize in chemistry each "for the development of soft desorption ionisation methods... for mass spectrometric analyses of biological macromolecules." 5 Malcolm Dole 1968 developed ESI technique for the first time 6 1

2 Nobel Prize in Chemistry for their developments of soft desorption ionisation methods for mass spectrometric analysis of biological macromolecules. John B. Fenn (USA) Virginia Commonwealth University Electrospray Koichi Tanaka (Japan) Shimadzu. Corp. Kyoto Laser Ionization HOW DOSE A MS INSTRUMENTS WORK? 7 8 Physics of a MS instrument Mass Spectrometry (MS) is an analytical technique to accurately measure the mass of different molecules within a sample Types of MS Components ION SOURCE ESI : Electrospray Ionisation MALDI : Matrix Assisted Laser Disorption/Ionization 11 MASS ANALYZER Quadrupoles Ion traps Time-of-flight with reflectron TOF/TOF FT-ICR : (Fourier transform Ion Cyclotron Resonance) QqTOF DETECTION (followed by database analysis) Faraday cup Scintillation counter Electromultiplier High-energy dynodes with electronmultiplier Array (detector) FT-MS 12 let's watch a video 2

3 INTERPRETATION OF MS DATA (A TYPICAL MASS SPECTRUM) A typical mass spectrum a of peptide chain Effect of fragmentation Isotopes effect The mass spectrum of water m/z =18 (H20) since O H H = = 18 amu m/z=17 (HO) since O H = 16 1 = 17 amu m/z=16 (O) since O = 16 amu m/z=1 (H) since H = 1 amu calculation of rubidium molecular weight Soft ionization techniques keep the molecule of interest fully intact Heated Incandescent Tungsten/Rhenium Filament DESIGN TO REDUCE COMPLEXITY HELPS BIO- MACROMOLECULES TO BE ANALYZED e 70 ev

4 MALDI Matrix Assisted Laser Desorption/Ionization (MALDI) MALDI TOF/TOF MASS SPECTROMETERS Method where a laser is used to generate ions of high molecular weight samples, such as proteins and polymers. I. Sample (A) is mixed with excess matrix (M) and dried on a MALDI plate. II. Laser flash ionizes matrix molecules. III. Sample molecules are ionized by proton transfer from matrix: MH A M AH HT Spotting on a MALDI Plate Crystal formation on MALDI plate How ionization occurs? The mechanism remains uncertain It may involve : I. absorption of light by the matrix II. transmission of this energy to the analyte III. Ionization of analyte into the gas phase as a result of the relatively large amount of energy absorbed To accelerate the resulting ions into a flight-tube in the mass spectrometer they are subjected to a high electrical field. MALDI -TOF Time Of Flight (TOF) TOF is a kind of mass analyzer TOF works just its name sounds TOF measures the time it takes for ions to fly from one end of the analyzer to the other and strike the detector The speed of flight is proportional to the ion s m/z values

5 Principle of MALDI-TOF-MS Vacuum lock all ions with E kin = 1/2mv 2 Vacuum system Linear flight tube Problems of linear mood of TOF analyzers The problem: Peaks are inherently broad in MALDI- TOF spectra (poor mass resolution). The cause: Ions of the same mass coming from the target have different speeds. This is due to uneven energy distribution when the ions are formed by the laser pulse. Sample matrix on target 25 Sample plate Analyte molecules in matrix Acceleration grids 20 to 200 spectra have to be added; total duration 2 to 20 seconds with 50 (200) Hertz Laser Drift region Ion detector Mass spectrum space/energy uncertainty Flight time m/z 26 Ions of same mass, different velocities Can we compensate for the initial energy spread of ions of the same mass to produce narrower peaks? Delayed Extraction/extraction potential Reflector TOF Mass Analyzer Delayed Extraction (DE) improves performance 0 V. Ions of same mass, different velocities Step 1: No applied electric field. Ions spread out. 0 V. 20 kv. 0 V. Step 2: Field applied. Slow ions accelerated more than fast ones. 20 kv. Step 3: Slow ions catch up with faster ones. 0 V What is a reflector TOF analyzer? Reflectrons are ion optical devices that reverse the direction of travel of an ion (reflect) in a mass spectrometer. A single stage gridded ion mirror that subjects the ions to a uniform repulsive electric field to reflect them. A reflector focuses ions to give better mass resolution Detector Ion Source Reflector (Ion Mirror) The reflector or ion mirror compensates for the initial energy spread of ions of the same mass coming from the ion source, and improves resolution V. 20 kv 5

6 High resolution TOF-MS with Reflector Reflector 0V kv MALDI ion source Ion detector Ion reflector The reflector focuses ions of same mass but different E kin (velocity) on detector; high resolution is obtained HiRes mass spectrum Flight time m/z 32 Tandem mass spectrometry (MS n ) Tandem Mass Analyzers & MALDI TOF/TOF INSTRUMENTS Tandem mass spectrometry (MS/MS) is used to produce structural information about a compound by fragmenting specific sample ions inside the mass spectrometer and identifying the resulting fragment ions. A tandem mass spectrometer is a mass spectrometer that has more than one analyzer, in practice usually two MALDI TOF/TOF as a tandem mass analyzer MALDI can also be used for tandem mass spectrometry studies in combination with two consecutive TOF tubes that are separated by a collision cell. The ion fragment selected from the first TOF tube is further fragmented in the collision cell by bombarding against an inert gas like argon. This technique can be used for protein sequencing studies. Laser Detect or PEPTIDE MASS FINGERPRINTING & PEPTIDE SEQUENCING TOF 1 TOF 2 Collision cell Reflector

7 % Intensity Reflector Spec #1 MC=>TR[BP = , 15779] Mass (m/z) E4 5/18/2014 Peptide Mass Fingerprinting (PMF) Peptide Mass Fingerprinting (PMF) Used to identify protein spots Depends of the fact that if a peptide is cut up or fragmented in a known way, the resulting fragments (and resulting masses) are unique enough to identify the protein Requires a database of known sequences Uses software to compare observed masses with masses calculated from database Database searching Effect of Mass Accuracy on PMF search results Tryptic digest of human HB-alpha chain yields 14 tryptic peptides The peptide VGAHAGEYGEALER has the exact mass of Da Peptide Sequencing Mass based Peptide Sequencing Helps decoding of structural information & amino acid sequences Use of Tandem MS is inevitable Needs special algorithms for mining specific features of Tandem MS data (SALSA)

8 Peptide Sequencing by MS/MS N2 inert Gas CID and peptide fragmentation Q0 Q1 Q2 Q3 MS - Peptide Mass Fingerprint MS/MS - Peptide tag Schematic representation of nomenclature for fragmentation of peptide ions Possible b- and y-ion fragments for the peptide AVAGCAGAR. Structures of the b4 and y5 ions from cleavage between the glycine and cysteine residues are depicted TOP-DOWN VS. BOTTOM-UP PROTEOMICS Ala CID and peptide fragmentation Annotated MS-MS spectrum of [M2] 2 ion of AVAGCAGAR showing b- and y-ions Top-down proteomics starts with intact proteins to identify and characterize many proteoforms. Top-down proteomics strategies The name top-down comes from the fact that the analysis starts from the top using the intact protein. The approach skips the digestion step and instead puts intact proteins directly into the mass spectrometer, where the instrument breaks them down into smaller fragments. 47 By starting with intact proteins, rather than their pieces, top-down analysis more accurately reflects the structure and properties of actual biological systems than does bottom-up proteomics. 48 8

9 Bottom-down proteomics Bottom-up proteomics strategies Bottom-up strategies are most potent delivering sequence information from: low abundant protein components of complex mixtures. from extraordinarily hard-to-handle proteins. Important approaches to bottom-up analysis: sort, then break, as achieved by 2-D gel electrophoretic separation, ingel tryptic digestion, LC MS/MS and break, then sort achieved by first cleaving proteins within a solubilized lysate followed by multidimensional chromatography interfaced to data-dependent tandem mass spectrometry (i.e., multidimensional protein identification technology (MudPIT), or shotgun proteomics) Top-down vs. Bottom-up proteomics strategies Questions? Need More Information? Feel free to contact us. Neda Saraygord-Afshari PhD afshari,.edu@gmail.com