An Introduction to the ProteinChip® Reader

Matrix-Assisted Laser Desorption/Ionization (MALDI) Samples introduced to the MS on a passive probe Desorption/ionization method for non-volatile compounds analyte embedded in a solid-state matrix crystal Matrix - energy absorbing compound responsible for converting laser energy to thermal energy, which facilitates the desorption/ionization process Discovered in late 1980s by groups in Germany and Japan KOICHI TANAKA SHARES 2002 NOBEL PRIZE FOR APPLICATION OF LASER DESORPTION TO LARGE BIOMOLECULES

Surface Enhanced Laser Desorption/Ionization (SELDI) Desorption/ionization method of non-volatile compounds in which the sample presenting surface plays an active role in the extraction, presentation, structural modification, amplification, and/or ionization of a given sample Invented by T. William Hutchens and Tai-Tung Yip in early 1990s

The SELDI-TOF Process Matrix Crystals Embedded Proteins Chip Surface

Matching EAM with Sample Important EAM characteristics High molar absorptivity at laser wavelength Co-crystallizes well with sample Yields peaks that do not interfere with sample spectrum Does not promote multiple protonation Commonly used EAMs CHCA (α-cyano-4-hydroxycinnamic acid) Matrix ions generally < 600 Da Promotes multiply protonated protein ions SPA (sinapinic acid) Higher m/z matrix ions Less multiply protonated protein ions EAM-1

The Time-Of-Flight Mass Spectrometer 20 KV Acceleration Region Free Flight Region (Drift Tube) X1 X2 t1 t2

Time-Of-Flight Equation E = 1/2 mv2 = z · V 2V ∆x x1 + x2 m/z = where v = = v2 ∆t t1 + t2 2V m/z = · ∆t2 ∆x2 2V Assuming is constant, ∆x2 V= acceleration voltage v= average final velocity m/z = K · ∆t2

Ion Populations and Interpretation of Mass Spectra Parent molecular ion: formed by adding a charged species (usually a proton) (M+H)+ Pseudomolecular ion: formed by adding multiple protons; an ion formed by adding single or multiple non-proton charging species; an ion formed by clustering of parent molecular species; or a combination of the latter (M+2H)+2, (M+Na)+, (2M+H)+, (2M-H+2Na)+

Interpretation of Mass Spectra M/z: the mass-to-charge ratio M/z = MWanalyte + S(MWcs) S Charge cs = charging species

Time-Of-Flight Fundamentals Detector Field Free Region Ion Source Time Ions leaving the source are accelerated to the same energy. Ions of different masses have different velocities, and therefore arrive at the detector at different times

Resolution Resolution is the ability to create separate signals from ions of similar mass Analogous to chromatographic efficiency Expressed as m/∆m and equals t/2∆t Resolution is unitless Resolution = peak centroid/FWHM H H/2 WH 1/2

Time Lag Focusing: Lag Period Time Lag Focusing: Ion Extraction Period V1 Repeller Source Lens Ext.. Lens Gnd. Image Plane Time Lag Focusing: Lag Period s x V2 Time Lag Focusing: Ion Extraction Period Ext. Potential S position d

Ion Deflection – prevents detector overload Deflector Plates source block sample post + ProteinChip array What Time Lag Focusing does: Time lag focusing improves mass resolution at a specific “focus mass” which is chosen by the User. Resolution – the problem: Peak resolution deviates from the ideal for a number of reasons. One of the contributing factors is that, following irradiation with the laser, molecules of the same mass desorb with different velocities. Time lag focusing addresses this problem. Time lag focusing – the solution: TLF tightens the ion cloud at a specified mass by providing molecules with higher desorption velocities proportionately less additional kinetic energy as they travel through the ion optics, so those which desorbed at lower velocities catch up to the others as they reach the detector. In other words, time lag focusing works by providing the slower ions with extra kinetic energy, thus speeding them up, so they can catch up with the faster molecules when the group of ions reaches the detector. Caveats: One limitation of TLF is that it is not possible to focus on a wide mass range all at once. In other words, in order to sharpen peaks from a spectrum at 2 kDa and at 15 kDa, data must be collected on two different occasions, with two different TLF settings. Moreover, the resolution improvement from TLF falls off rapidly as mass increases beyond 20 kDa. Finally, as a rule of thumb when analyzing a broad mass range, it is better to “under-focus,” focusing on a mass at the low end of the range, rather than to “over-focus.” HV contact

Deflecting ions at low mass Extractor Repeller Ground Ion Detector Deflectors + 0 V 0 V applied at td - V - V applied from t0 to td

Effect of Ion Deflector setting on peak detection 2500 5000 7500 Deflector 0 Deflector 1000 Deflector 2000 Deflector 4000 Deflector 8000 10 20 30 1084.8+H 1638.6+H 3496.0+H 5806.3+H 7029.7+H 40 50 1084.6+H 1638.7+H 3495.8+H 5805.5+H 7029.1+H 5 15 1621.4+H 3495.5+H 5805.3+H 7028.5+H 3491.0+H 7028.9+H 2.5 7.5 7007.4+H

Multiple Stage Electron Multiplier Ion Detector Input Ions First Dynode (ion impact surface) Electrons Emitted from 1st Dynode 2nd Dynode 3rd Dynode HV Detector Output Dynode Chain

The Effects of Mass and Accelerating Voltage on Sensitivity Increasing Sensitivity Increasing Accelerating Voltage

Linear LDI TOF MS TLF CIE PIE Extractor Repeller Ground Laser Lens Mirror Attenuator Beam Splitter Trigger Photo Diode High Voltage PS HV Ion Extractor Delay Generator Software Processing Ion Detector CIE TLF PIE Linear LDI TOF MS High Speed A/D

Molecular Weight Accuracy System calibration Well-characterized samples of known molecular weight are analyzed and their flight times used to derive the coefficients for a TOF calibration function on a specific instrument Calibrations are best performed using identical or similar compounds and/or matrices Multi-component mixtures providing multiple calibration points should be used (at least 3 points) Multiply-charged or cluster ions have different total kinetic energies when compared to their parent molecular ions

Molecular Weight Accuracy (Cont.) Internal standard measurement (“internal calibration”) System calibration is performed during analysis of the unknown Provides the highest degree of MW determination accuracy Calibrants are added to unknown sample in an effort to bracket the unknown with calibration points Ion suppression may occur External standard measurement (“external calibration”) System calibration is performed as a separate experiment Typically 5–10X increase in error Easiest to perform on unknown mixtures

Mass Calibration and Initial Kinetic Energy Molecular Wt (Da) Low MW Calibration Hi MW Calibration MW Error Square of Flight Time (uSec2)

Calibration Errors: Extrapolation vs. Interpolation Calibrant Average MW Calculated MW (using all 5 standards) Calculated MW (using 3 smallest) Calculated MW (using 3 largest) Cytochrome C, horse 12360.2 12345.4 (-1198 ppm) 12361.5 (+105 ppm) 12296 (-5194 ppm) Myoglobin, horse muscle 16951.5 16942.8 (-513 ppm) 16949.5 (-118 ppm) 16891 (-3569 ppm) GAPDH, rabbit 35688 35733 (+1261 ppm) 35689 (+28 ppm) 35686 (-56 ppm) Albumin, bovine serum 66433 66459 (+391 ppm) 66312 (-1821 ppm) 66438 (+75 ppm) Β- Galactosidase, E. Coli. 116351 116304 (-404 ppm) 115971 (-3266 ppm) 116348 (-26 ppm)

Summary of Spot to Spot Calibration for an 8 Spot ProteinChip Array

Mass variation without Spot to Spot Calibration CVMASS = 0.046%

Mass variation with Spot to Spot Calibration CVMASS = 0.036%

SEND – Matrixless Ion Production Surface Enhanced Neat Desorption First Product – SEND C18 Primary Application is Peptide Mass Fingerprinting for Protein Identification Superior sensitivity over current arrays (NP20 & H4) Less background from matrix peaks (especially above 600 Da) Competitive Technology to C18 ZipTips C18 functionality chosen to allow desalting of digest on-chip Ability to use less sample compared to ZipTip clean-up method

Sensitivity Comparison: C18 SEND vs CHCA Amount of digest Number of BSA peptide detected by C18 SEND Number of BSA peptide detected by CHCA on NP20 Percent coverage of BSA Sequence by C18 SEND Percent coverage of BSA Sequence by CHCA on NP20 1000 fmol 38 40 61% 62% 100 fmol 39 37 59% 10 fmol 27 13 47% 26% 1 fmol 15 35% 0% At <100 fmol SEND shows improved sensitivity over NP20/CHCA

Sensitivity Comparison at 10 fmol C18 SEND CHCA

Sensitivity Comparison at 1 fmol C18 SEND CHCA

Sample Clean up Comparison: C18 SEND vs C18 ZipTip Amount of digest Number of BSA peptide detected by C18 SEND Number of BSA peptide detected by C18 ZipTip Percent coverage of BSA Sequence by C18 SEND Percent coverage of BSA Sequence by C18 ZipTip 1000 fmol 23 38 46% 59% 100 fmol 19 25 40% 44% 10 fmol 16 3 34% 6.3% Advantage of SEND is seen at <100 fmol level (this is a common range for users to work in). At high concnetration ZipTip has an increased capacity compared to SEND hence the better performance.

Sample Clean Up Comparison at 100 fmol C18 SEND, 100 fmol C18 ZipTip, 100 fmol

Sample Clean Up Comparison at 10 fmol C18 SEND, 10 fmol C18 ZipTip, 10 fmol