1. An Introduction to the ProteinChip® Reader

2. 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

3. 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

4. The SELDI-TOF Process
Matrix Crystals
Embedded Proteins
Chip Surface

5. 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

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

7. 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

8. 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)+

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

10. 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

11. 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

12. Time Lag Focusing: Lag Period Time Lag Focusing: Ion Extraction PeriodV1
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

15. 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

16. 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

19. Effect of Ion Deflector setting on peak detection
2500
5000
7500
Deflector 0
Deflector 1000
Deflector 2000
Deflector 4000
Deflector 8000 10 20 30 H H H H H 40 50 H H H H H 5 15 H H H H H H
2.5
7.5 H

20. 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

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

22. 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

23. 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

24. 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

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

26. 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
(-1198 ppm)
(+105 ppm)
12296
(-5194 ppm)
Myoglobin, horse muscle
(-513 ppm)
(-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)

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

28. Mass variation without Spot to Spot Calibration
CVMASS = 0.046%

29. Mass variation with Spot to Spot Calibration
CVMASS = 0.036%

30. 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

31. 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

32. Sensitivity Comparison at 10 fmol
C18 SEND
CHCA

33. Sensitivity Comparison at 1 fmol
C18 SEND
CHCA

34. 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.

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

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