Download Introductory Signposting - Oklahoma State University

MALDI-TOF Mass Spectrometry and
Introduction to Proteomics
Dr. Steve Hartson
Oklahoma State University
Dept. Biochemistry and Molecular Biology
Recombinant DNA/Protein Resource Facility
“Since its introduction more than 100 years
ago, mass spectrometry has become a ubiquitous
research tool. The number of scientific
discoveries that MS enabled and the number of
Nobel Prizes awarded to scientists who
contributed to the method’s evolution is
astounding. Used extensively by chemists,
biologists, pharmacologists, and scientists in
several disciples, MS how holds a prominent spot
in the world of analytical techniques.”
-Tanuja Kopal
Editor in Chief
Genomics & Proteomics
“Astounding prizes!”
Joseph John Thomson
1906 Nobel Prize for Physics
"in recognition of the great merits of his theoretical and experimental investigations
on the conduction of electricity by gases”
Francis William Aston
1922 Nobel Prize for Chemistry
"for his discovery, by means of his mass spectrograph, of isotopes, in a large
number of non-radioactive elements, and for his enunciation of the whole-number
rule”
Wolfgang Paul
1989 Nobel Prize for Physics
"for the development of the ion trap technique”
John Bennet Fenn
2002 Nobel Prize for Chemistry
"for the development of soft desorption ionisation methods (ESI) for mass
spectrometric analyses of biological macromolecules”
Koichi Tanaka
2002 Nobel Prize for Chemistry
"for the development of soft desorption ionisation methods (MALDI) for mass
spectrometric analyses of biological macromolecules"
MS is a leading biotool
Session subjects at the 2004 meeting of the
Association of Biomolecular Resource Facilities
9
8
7
6
5
4
3
2
1
0
sequ
frags
array
RTPCR
siRNA
other
MS
peptides
structure
DNA
Protein
Bioinform
Other
molecules
OK, how about modern times?
• In the 2005 ABRF meeting, mass spectrometry
and proteomics were given their own satellite
meeting on a separate day.
• In 2006, it was business as usual, with MS and
proteomics comprising 45% of the presentations;
no other subjects came close.
MALDI-TOF mass spectrometer
MALDI Ion source
TOF Mass Analyzer
“MALDI-TOF” mass spec
• Matrix-Assisted:
Your favorite
molecule is cocrystallized (dried)
with a light-adsorbing
compound (“matrix”).
“MALDI-TOF” mass spec
• Laser
Desorption/Ionization
: A pulse of laser
light is used to force
molecules into gas
phase and ionize
them.
+
+ + +
+
+
+
MALDI-TOF mass spectrometry
• Ions are then
accelerated in a highvoltage electrostatic
field…
(-)
++ + +
+
+ +
(+)
MALDI-TOF mass spectrometry
…and allowed to drift
down a long highvacuum flight tube.
• At the end of the
tube, ions strike a
detector.
• Drift time (time of
flight) is measured by
sophisticated
electronics.
+ + + +
+
+ +
(-)
Interpreting a mass spectrum
• x-axis is…
– tof
–m / z
– “mass” of ion
• y-axis is…
– “hits” on the
detector
– number of ions
detected
TOF~velocity~acceleration~mass
• The relationship of TOF v.s. mass is
calibrated via co-analysis of standards
whose masses are known.
More spec’s...
• Exacting determination of masses
(20 to100 ppm mass accuracy)
• Exquisite sensitivity
(routine detection sub-pmoles of peptide)
Mass Spec Concepts and Vocab
• “ions”
• “mass”
• “isotopes”
Ions
• Ion = “charged molecule”
• 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)
Isotopes
• Stable natural
isotopes
– extra neutrons
versus the
periodic table
– not radioisotopes
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.
Overview of a typical protein
identification by MALDI-TOF
• Isolation of protein(s) of interest
• Cleavage at specific residues
• MALDI-TOF analysis of resultant
peptides
• Extract peptide masses from the spectrum
• Search databases for matching “peptide
mass fingerprint”
Cleavage Fingerprinting to
establish protein identity
Cleveland mapping: after
cleavage with a
specific protease,
peptide products are
compared in adjacent
lanes on an SDS-PAGE
gel.
PMF: each MS peak is represents
the peptide product of a cleavage.
Spec #1 MC[BP = 1053.6, 44590]
1053.6285
100
4.5E+4
656.1051
90
869.4992
80
70
% Int e ns it y
60
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
1890.0891
591.3327 741.4249
1569.9111
893.4636 1075.6256
585.3485
1443.
8
027
537.3699 679.4092
1088.6108 1278.6373
1674.6634
800.4719 919.5099
1252.6669 1436.7851 1625.0525
1003.8684
1432.1174
527.2609
760.6
1156.2
1551.8
Mass (m/z )
2008.2726
1893.1110
2033.1769 2185.2870
1856.6127
1947.4
0
2343.0
A “peptide mass” fingerprint
Spec #1 MC[BP = 1053.6, 44590]
1053.6285
100
4.5E+4
• "Peaks" represent
individual peptides.
656.1051
90
869.4992
80
• A "peak list"
representing these
peptides' masses can
be generated and
used.
70
% Int e ns it y
60
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
919.
5
099
800.4719
1003.8684
527.2609
760.6
1890.0891
1569.9111
1278.6373 1443.8027
1252.6669 1436.7851
1432.1174
1156.2
1551.8
Mass (m/z )
1674.6634
1625.0525
2008.2726
1893.1110
2033.1769 2185.2870
1856.6127
1947.4
0
2343.0
Experimental proteins
v.s.
Hypothetical proteins
• Digest experimental
protein with protease
• Size resultant peptides
• Generate list of “peak
masses” (a.k.a.,
peptide mass
fingerprint)
• Archive of protein sequences
• “Index” via in silico digestion
with protease
• Calculate theoretical masses of
individual “peptides.”
• Generate a “look up table” for
use by a scoring algorithm to
allow ranking of “matches.”
At this point in the technology, the database is
not built from experimental mass spec data!
•
•
•
•
Logistics
Five Units / Four groups
Rotation as per page 3
Lecture Mon morn, Thurs morn
Thurs & Friday by reservation
final Unit: assessing your samples
mopping up
• “Sticking points”
Voyager U1
Sample prep (U4B)