Download Overview of a typical protein identification by MALDI-TOF

Lecture Three: "MALDI-TOF
MS, Up Close, and Personal
• Review of principles
• Delayed Extraction
• Reflector / reflectron
• Post-source Decay (PSD)
Mass Spec vocabulary
• Generic mass spectrometer
• Ions & isotopes
• Mass:
–
–
–
–
m/z
monoisotopic mass
average mass
peak centroid
• Resolution
Ions
• Only ions are detected in MS
• For ionization techniques that are typically
used for biological molecules, ions are
generated via the ejection or capture of a
proton.
• The mass of a proton is ~1 AMU; the charge
of a proton is +1
• Ionic mass [MH]+1 versus molecular mass
(subtract ~1 from raw data)
Peaks
• A peak represents a
packet of peptide ions
hitting the detector.
• The distribution of ions’
flight times creates a
rapid rise, and then fall,
of current from the
detector (y-axis).
• This current is captured
digitally.
• Base Peak (BP): the
strongest peak in the
spectrum
Resolution: the MS gold standard
• Limits mass accuracy
• R=mass/peak width
•
•
•
•
R=1000
R=3000
R=10,000
R=30,000
Measuring Resolution
• Analyte mass divided by Full peak Width,
as measured at one-Half peak's Maximum
height (FWHM)
• Industry standard, despite being somewhat
arbitrary
• Alternatively, the DE-PRO can resolve
analytes with a difference of 1 part per
thousand (linear mode)…
…or a difference of 1 part per 6 thousand
(reflector mode).
monoisotopic mass,
average mass,
peak centroid
high-resolution spectrum
with isotopes resolved
low-resolution spectrum with
unresolved “isotopic envelope”
Mass versus m / z
• Mass divided by
charge
• +1 ions, +2 ions,
+3 ions,…
+20…+30.
• "twice the charge"
behaves like "half
the size"
Isotopes
• For peptide-sized
molecules, most mass
spec’s can resolve (n)
versus (n +1 AMU).
• The result is that a
single peptide actually
yields a series peaks
differing by one AMU.
Calibration and mass error
• MALDI-TOF’s must be rigorously
calibrated, due to TOF variance across the
face of the probe plate.
• Other MS’s need less frequent calibration.
• You will always have error
– error as a pitfall
– error as a tool
Drift time allows mass determination
because:
drift time~velocity~ acceleration~mass.
The measurement is calibrated by co-analysis
of standards whose masses are known.
TOF ~ m / z
Spec #1 MC[BP = 1053.6, 44590]
100
4.5E+4
656.1051
90
869.4992
80
70
60
% Int ensit y
549.3439
617.4088
822.4886
845.5043
869.4992
916.5234
947.5222
1036.598
1053.629
1072.603
1101.621
1117.628
1355.756
1426.866
1438.867
1440.831
1053.6285
1117.6282
50
549.3439
822.4886
1072.6033
1550.8736
1355.7562
40
1426.8657
1690.9995
617.4088
1566.8917
30
916.5234 1036.5975
20
10
0
365.0
591.3327
741.4249
893.4636
585.3485
1075.6256
537.3699 679.4092
1088.6108
800.4719 919.5099
1003.8684
527.2609
760.6
1890.0891
1569.9111
1278.6373
1252.6669
1156.2
1443.8027
1436.7851
1432.1174
1551.8
Mass (m/z)
1674.6634
1625.0525
2008.2726
1893.1110
2033.1769
1856.6127
1947.4
2185.2870
0
2343.0
Calibration
• drift
time~velocity~
acceleration~mass.
• The relationship
between TOF and
mass can be
calibrated using
standards with
known masses.
• …or "default"
estimates.
7000
6000
5000
4000
3000
2000
1000
0
100
200
300
400
500
600
Calibrations must follow the laws
of physics
• drift
time~velocity~
acceleration~mass.
• This relationship is
linear, and major
departures are not
physically possible.
• NONSENSE -->
4500
4000
3500
3000
2500
2000
1500
1000
500
0
100
200
300
400
500
600
700
Calibration
• drift
time~velocity~
800
acceleration~mass.
700
• In a calibration
600
effort and in a
database search
500
result, errors
400
between data and
300
theory must be
200
systematic and/or
within instrument
100
tolerances.
0
1000 2000 3000 4000 5000 6000 7000
theory
acceptable
still OK
nonsense
Calibration and Mass Error
• In PMF d-base search results, differences between
theoretical mass and experimental mass arise from two
causes:
– Calibration error
– An invalid, coincidental match between your data and the
theoretical protein
Mass Error
Calibration
• drift
time~velocity~
800
acceleration~mass.
700
• In a calibration
600
effort and in a
database search
500
result, errors
400
between data and
300
theory must be
200
systematic and/or
within instrument
100
tolerances.
0
1000 2000 3000 4000 5000 6000 7000
theory
acceptable
still OK
nonsense
Fractional Mass as a tool
• Although 12C is 12.0000 AMU, other atoms have a
decimal component which is not zero-a fractional
mass.
• This fractional mass contributes to peptide mass in
a consistent manner: ~ 0.5 Da per kDa.
• This consistent trend can be used to assess
calibrations: if the FM is wrong, the calibration is
suspect.
• This trend is a powerful way to ID artifacts in
peak lists (matrix, de-isotoping errors, noise).
powefu
Lecture Three: "MALDI-TOF
MS, Up Close, and Personal
• Review of principles
• Delayed Extraction
• Reflector / reflectron
• Post-source Decay (PSD)
Practical MALDI considerations
• You need crystals of peptide:matrix.
• You’ll have matrix noise, especially:
– When signal is low;
– In the low-mass range
• Ionization is a competitive process:
– minimal matrix, salt, trypsin
The problem:
• The desorption process imparts intitial
velocities to analyte molecules (independent
of, and prior to, the accelerating voltage).
• These initial velocities are not uniform;
they have a significantly wide range.
• These non-uniform initial velocities are
significant and affect TOF.
• The result is broad TOF peaks of analytes
with identical masses (poor resolution).
The solution:
• Exploit the initial velocities by delaying the
“extraction” (the application of the accelerating
voltage).
• Delay allows the initial velocities to be translated
into distance from the plate.
• When the plate is charged, this distance will
impact the time spent in the accelerating field.
• Time spent in the accelerating field will impact
the magnitude of acceleration (and thus,
velocity/TOF).
The skate park analogy:
Identical twins on skate boards
Have the same mass...
“Go!”
…but the green skater cheats;
he hits the board running, and thus
has a greater initial velocity...
…which will allow him to pull
ahead of his twin, despite gravity’s
equal acceleration of both skaters...
“And they’re off…”
…thus, despite their equal masses,
the skaters will not hit the finish line
(the “detector”) at the same time.
The solution...
“Go!”
(Again, our identical twins have
the same mass but different
initial velocities…)
Use a slight delay to translate the initial
velocity into distance before applying the
acceleration...
And they’re off!
(No “slope” = no electrostatic field applied to accelerate the ions.)
…then apply the accelerating field.
The purple skater will experience
more acceleration than the green skater.
This greater acceleration of the formerly
slow ion results in a greater velocity
during the time of flight...
…allowing the purple skater to catch up...
…and the two skaters of equal mass reach
the finish line (the detector) simultaneously,
despite having different initial velocities.
We have focused the
arrival of our twin
analytes at the detector.
One last twist: grid voltage
We break the accelerating voltage into two
slopes using the grid.
This gives us more control
in our manipulation
of time lag focusing.
Delay time and Grid
voltage are
interdependent parameters.
Lecture Three: "MALDI-TOF
MS, Up Close, and Personal
• Review of principles
• Delayed Extraction
• Reflector / reflectron
• Post-source Decay (PSD)
The reflector is an “ion mirror”
that redirects the vector of ion
flight.
Reflectors dramatically increase
resolution...
• …and by creating a slightly longer flight
path (greater separation between peaks)…
• …by focusing the arrival of ions having the
same mass, but slightly different velocities
(sharper, narrower peaks.
MALDI-TOF Theory-overview
•
•
•
•
•
Our instrument
General theory of MALDI-TOF
Delayed Extraction
Reflector
Post-source decay (PSD)
Post Source Decay
• PSD is a non-specific cleavage tool that can
be used to generate fingerprints of
individual peptides.
• Referred to as an “MS/MS” technique
because ions are re-accelerated via the
reflector.)
• CID and CAF can be used to facilitate
decay.
Peptides decay (break) during flight.
Breakage generates characteristic
products (and nomenclature).
The masses of daughter ions can
be determined.
• TOF ~acceleration~mass
• First acceleration: from the source
• Second acceleration: reflection in the
mirror.
Only ions with a specific TOF (the
parent ion and its daughters) are
allowed to enter the ion mirror.
• Too many analytes!
• A specific peak mass
range is chosen by the
operator.
• “Timed ion selector”
(Bradbury-Neilson
gate)
Problem: daughters don’t arrive
on time
• No single reflector voltage (“mirror ratio”
relative to accelerating voltage gradient) will
properly focus all daughter ions.
• Although all fragments from a single parent
have the same velocity...
light daughter ions have less energy, and
will be reflected too easily.
heavy daughter ions have more energy,
and may pass through the ion mirror.
The solution:
• Use a slightly lower “mirror ratio” (reflector
voltage) for each daughter size range.
• “Mirror ratio” is the reflector’s voltage
relative to the accelerating field.
At mirror ratio 1.00...
Parent MH+ (1000 Da ) is properly focused.
Daughter AH+ (700 Da) is poorly focused.
Daughter BH+ (300 Da) is poorly focused.
At mirror ratio 0.7 (a shallower
voltage gradient)...
Parent MH+ (1000 Da) is not reflected.
Daughter AH+ (700 Da) is properly focused.
Daughter BH+ (300 Da) is poorly focused.
At mirror ratio 0.3 (an even
shallower voltage gradient)...
Parent MH+ (1000 Da) is not reflected.
Daughter AH+ (700 Da) is not reflected.
Daughter BH+ (300 Da) is properly focused.
The result is several “mini-spectra”
which are “stitched” together to
yield a composite spectrum.
In the composite spectrum...
• The heaviest peak represents the parent (this
intense peak may need to be cropped off for
display purposes).
• Other peaks represent decay products.
Uses of PSD
• Confirmation tool
• Can be used for searching.
• Analysis of labile post-translational
modifications.
• CAF
• Fragmentation of organic.
Limitations
• Efficiency of breakage
• Mass accuracy
• Our manufacturer:
– no automation=tedious
– Their corporate investments focused on MS/MS
End of Lecture Three:
• Questions?
• Next up: "Bigger, Better, Faster, Stronger:
the Cutting Edge"