Download Metalloprotein/Metallocofactor Mass Spectrometry

Metalloprotein/Metallocofactor Mass Spectrometry
Jeffrey N. Agar
Brandeis University
Diseases Involving Transition Metals
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Hereditary hemochromatosis Type 1‐4
Aceruloplasminemia
Hypotransferrinemia
Menkes disease
Wilson disease (autosomal recessive)
ALS‐like symptoms in mice
Antibiotic resistance plasmids in bacteria ALS
Fatal Neonatal Lactic Acidosis
Fatal deficiency of multiple respiratory functions
dehydrogenase enzymes
Swedish myopathy with exercize intolerance
Fredrich ataxia Sideroblastic anemia
Defect mitochondrial heme biosyn and COX def.
Neurodegeneration (AD, PD, HD, Prion, etc)
HFE, ?, TFR2, Ferroportin, respectively. Ceruloplasmin (CP)
Transferrin (TF)
MNK (ATP7A)
WD (ATP7B)
IRP
Heavy metal transporters (MERP, etc)
SOD1
C12orf62
NFU1 and BOLA3 cause a and 2‐oxoacid ISCU FXN
GLRX5
COX10, COX15 (fatal cardiomyopathy)
Mitochondrial dysfunction
This is not an exhaustive list email [email protected] if you know of more…
Goal of Lecture
• Learn fundamentals of mass spectrometry (MS) interpretation
• Learn fundamentals of mass spectrometry instrumentation in historical perspective
• Overview of MS applications
• Metalloprotein MS applications
Mass Spectrometry at the Interface of Physics, Chemistry, and Biology. Mass Spectrometer Basics
I. Ion Source (makes ions out of neutrals).
II. Mass Analyzer (separates ions).
III. Detector (Black Box For This Class).
IV. Computer (Black Box For This Class).
Mass Spectrometer Basics
I. Ion Sources (the three most popular)
‐
‐
‐
Electrospray ionization ‐ THE ONLY METALLOPROTEIN FRIENDLY TECHNIQUE.
MALDI ionization
Electron Ionization
II. Mass Analyzers (the only four)
‐
‐
‐
‐
Magnetic Sector
Quadrupole Time‐Of‐Flight
Fourier Transform
Physical Concepts Basics
I.
II.
III.
IV.
Only charged particles are analyzed in MS.
Negative attracts positive
Magnets bend trajectory of MOVING charges
Mass spectrometers perform three basic tasks.
I. Weigh
II. Purify
III. Break (react)
MS Basics‐ Monoisotopic Mass versus Average Mass.
• Average Weight/Mass. – You are use to thinking in bulk terms (lots of molecules) about weight or mass. For example, if you purify a Mol of Cu, it will weigh 63.5463 grams. That’s Cu’s “standard molecular weight” or “molar mass” or “relative atomic mass” or “average mass.”
– Obviously, no single atom of Cu weights 63.54 Daltons (Da, or amu)‐ that would require having half of a nucleon. The molecular weight of Cu is the weighted average of ~ 69% 63Cu and ~31% 65Cu.
MS Basics‐ Monoisotopic Mass versus Average Mass.
• Monoisotopic Mass (not weight!). – In mass spec we can often resolve the isotopomer distribution. If we can, the monoisotopic mass, which is a type of molecular mass (a mass of a particular atom or molecule), is a better (more accurate) description.
– The picture to the right
illustrates the mass spectrum
of Cu.
http://www.webelements.com/copper/isotopes.html
MS Basics: The mass defect
• Carbon is by definition 12.000000.
• Using this standard, mass spectrometrist realized that no other atom mass ends in “.000000
• Nuclei that are more stable than carbon weigh a bit less (32S “weighs” 31.97 Da), and less stable weight a bit more (226Rn, with a few millisecond halflife, “weighs” 226.0309).
• This is the result of differences in the relativistic mass of the nuclear binding energy (E=mc2)‐ particularly the mass of photons released when a particular element is created. • The nominal mass is the number of nucleons.
• We can use the nominal mass in conjunction with the mass defect to infer molecular formula! For example a molecular mass of 720.000000 is C60, not C59H12 (720.084 Da).
MS Basics: Mass Defect
Mass Defect results from difference in binding energy per nucleon (E=mc2)
MS Basics: Mass Defect
Mass Defect results from difference in binding energy per nucleon (E=mc2)
MS Basics: Resolution
Resolving Power (m/z ∕ ∆ m/z) Resolution (∆ m/z) (full width of peak at half height, (FWHH or 10% of valley)
High resolution >10,000 resolving power, FTMS > 100,000
MS Basics: Molecular Formula From Mass
• One of the most useful things a mass spectrometer can be used for is to determine the formula (and often identity) of a compound using ONLY ITS ACCURATE MASS.
• If you measure a compound that weighs 26.0140 Da, what is its molecular formula?
• The key is using both the nominal mass (26) and the mass defect.
• Remember that C12 is DEFINED as mass 12.00000000000000000000000000000
• What are the mass defects of the two elements present in this formula?
• Upper mass limit of roughly ~600 Da (using an FTMS) for normal organic compounds
MS Basics: How to interpret a mass spectrum
1. Assign charge state‐ in high resolution MS this is done by determining the distance in m/z between two isotopes of a molecule. The reciprocal of this distance is the charge state. For low resolution data use MAXENT or a charge state ruler.
2. Multiply the charge state by m/z and you have determined the molecular mass of a given peak within the mass spectrometer.
3. Next subtract mass that is an artifact of ionization to determine the actual mass. Charges are added during ionization in the form of proton adducts. MALDI usually adds a single proton and ESI multiple protons. Be careful‐ metals carry charges and are a special case that most mass spectrometrists wouldn’t think to account for.
Basics – Calculate Mass
~674.350 m/z
MS Basics Applied to Metals
• Metals have huge mass defects, 63Cu = 62.9295989, making them easier to detect. Automated data processing programs wont account for metals.
• Metals have characteristic isotope abundances‐
whereas only 1% of Carbon is heavy, 31% of Cu is heavy (63Cu).
• Metals have characteristic nominal masses.
• Metals usually have charges‐ It is critical for data analysis that you manually account for these charges being holes (not protons). Data analysis programs are not equipped to. Failure will result in a 1 Da overestimation in molecular mass per metal charge (so 4 Da on SOD1).
A Brief History of MS Technology
# of Mass Spectrometry Pubs Per Decade
(decade ending in a given year,
e.g. 2010 represents years 2000-2010)
MS Technology: Discovery of the electron’s e/m (Nobel Prize Thomson)
MS Technology: Thomson- Aston’s
Parabola Mass Spectrograph. Nobel Prize
1922
MS Technology: Electrospray Ionization (ESI) enabled metalloprotein characterization
In the Words of John Fenn:
Nobel Prize for ESI 2002
Mass Spectrometry is the art of measuring atoms and molecules to
determine their molecular weight. Such mass or weight information is
sometimes sufficient, frequently necessary, and always useful in
determining the identity of a species… Clearly the sine qua non (essential
element) of such a method is the conversion of a neutral analyte molecules
into ions.. In recent years, the efforts of many investigators have led to new
techniques for producing ions of species too large and complex to be
vaporized without substantial, even catastrophic, decomposition.
MS Technology: Nanospray‐ more sensitive and gentler than ESI‐ better for metalloproteins
ABI’s Lab On A Chip
Advion’s Nanomate
Matrix Assisted Laser Desorption Ionization (MALDI): Not good for metalloproteins‐ Often OK for small metal clusters (<1000 Da)
Tanaka’s 2002 Nobel Prize
MS Technology: Nanospray and MALDI in action
MALDI Ionization
Nanoelectrospray Taylor Cone
Typical Sensitivities of Ionization Techniques
MS Technology: Nier Sector Mass Spectrograph
Nobel Prizes Thomson/Aston 1910, 1922
MS Technology: Cyclotron‐ Lawrence Nobel Prize. Predecessor of first Fourier transform (high res) mass spectrometry.
Why is MS Technology so advanced? Preparative MS: The Manhattan Project
Calutron magnet was 15 feet in diameter
Original cyclotron below with blue
background was a couple of inches.
http://www.mbe.doe.gov/me70/manhattan/im
ages/MassSpectrograph.jpg
MS Technology: The Manhattan Project
MS Technology: The Manhattan Project
Lawrence wondering
why all of the quarters
in his pockets keep
disappearing.
Q‐FTMS
Hans Dehmelt Nobel
Prize 1989
Agar Lab FTMS is a Quad+TOF+ICR
MS Applications:
-Quantify small molecules including metallocofactors
and artificial additives in food and humansoften uses triple quad as with small molecule lab
Ben Johnson, Canadian athlete
Furabazol, Anabolic steroid
MS Applications ‐Identify explosives, biohazards, radioactive materials, toxic chemicals.
MS Applications
‐Identify Chemical Composition =>Saturn Mission; Jupiter Mission (1982 Galileo); Comet Mission
MS Applications
‐Identify Chemical Composition (Saturn Mission)
Science 13 May 2005:
Vol. 308. no. 5724, pp. 982 - 986
DOI: 10.1126/science.1110652
Ion Neutral Mass Spectrometer Results
from the First Flyby of Titan
J. Hunter Waite, Jr., et. al.
MS Application: Compound ID Using Electron Ionization Fragmentation
Ketone
Major fragmentation peaks result from cleavage of the C-C
bonds adjacent to the carbonyl.
4-Heptanone
C7H14O
MW = 114.19
Fifteen Years experience in providing mass spec libraries
Over 600,000 measured and identified mass spectra
Nearly 4 times the size of the NIST 2002 collection
MS Applications: Proteomics using MS/MS
Product Ion MS/MS (MS2) The Basis For The Proteomics Experiment
31MonoAverage
letter
letter
isotopic
Amino Acid
Structure 647 (+2)
at
all
m/z
(+3) 1) Scan Q1 to get Intensity
mass
code
code
mass
= mass spectrum (MS Survey)
Alanine
Ala
A
71.03712 71.08
647 (+2)
C3H5NO
Amino Acid Masses
Intens.
x10 6
Intens.
x106
648.9
3+
433.5
3
4
649.4
3
2+
649.0
2
2) Lock Q1 to Isolate only m/z 647
156.10112
156.19
And
trap molecules
in Q2 (collision cell)
2
Arginine
C6H12N4O
Arg
R
649.9
1
1
650.4
Asparagine
C4H6N2O2
2+
464.0
Asn
N
114.04293 114.10
0
640
642
Angio_tune_cid_000002.d: +MS
667.9
513.4
0
200
400
Aspartic acid
C4H5NO3
e n s.
x1 0 5
6
Asn or Asp
5
Cysteine
C3H5NOS
4
600
800
Asp
1000
D
6 4 7 .4
115.02695 115.09
3) Scan
Asx
B Q3 to get Intensity at all m/z
= MSMS (MS2; Collision Induced
Dissociation
MS (CID; CAD);
Product Ion MS)
Cys
C
103.00919
103.14
7 8 4 .4
3
Glutamic acid
Glu
C5H7NO3
E
2
129.04260 129.12
5 3 4 .3
Glutamine
C5H8N2O2
4 2 6 .2
1
3 8 2 .2
1 0 2 8 .6
Gln
Q
9 1 3 .5
128.05858 128.13
8 7 0 .4
0
500
750
An g i o _ tu n e _ ci d _ 0 0 0 0 0 5 .d : +M S2 (4 3 2 .0 ) M a xRe s
1000
1250
644
646
648
650
652
Angio_tune_cid_000003.d: +MS
654
656
658
m/z
MS Applications: 60,000 Year Old Protein MSMS
The osteocalcin that became a star was extracted from fossilized bison bone,
Bison priscus, which was radiocarbon-dated back almost 60'000 years. Bison
priscus, more commonly known as the steppe bison, though now extinct is quite
well known thanks to prehistoric paintings in Paleolithic caves and fossils found
in permafrost.
Automated Proteomics: Mascot ID
Good for IP “pulldowns” of interacting proteins
Metalloprotein MS applications: Don’t digest metalloproteins if you want to detect metals
homogenize
Many post-translational modifications, including
metals lost following endoproteinase digestion.
digest
Structural information can be lost during
digestion, especially unexpected modifications
Metalloprotein MS Applications: High res
methods and algorithms for isotopic fine
structure detection
Substance P,
human neuropeptide
(C63H98N18O13S)
m = E / c2
A
A transformative
transformative effect
effect
because
because higher
higher magnetic
magnetic
field
field increases
increases the
the upper
upper
mass
of
observing
mass of observing isotopic
isotopic
fine
fine structure
structure
and
and isotopic
isotopic fine
fine structure
structure
is
is a
a powerful
powerful tool
tool for
for
elemental
elemental composition
composition
confirmation
confirmation
Karabacak, NM et. al. 2010 JASMS . Li., L. 2009 JASMS and 2010 RCM.
Metalloprotein MS Applications:
Metal Content By Mass AND Charge State Distribution
G85R SOD1- Causes ALS (left) WT SOD1 (right)
tens.
x107
G85R holo in pure w ater_000003.d: +MS, Deconvoluted (MaxEnt)
Single
metal
8
Intens.
x107
4
Deconvoluated
spectrum
6
AS holo in pure w ater_000001.d: +MS, Deconvoluted (MaxEnt)
Deconvoluated
spectrum
holo
3
4
2
Single
metal+98
Da
2
1
holo
apo
0
15825
Single
metal
apo
0
15850
15875
15900
15925
15950
15975
16000
16025
m/z
15740
15760
15780
15800
15820
15840
15860
15880
15900
15920
m/z
Metalloprotein MS Applications:
Compare Experimental and Simulated Spectrum
Intens.
x108
15906.779
1.0
Red trace is experimental A4V
spectrum and black trace is
simulated spectrum w/ Cu/Zn
each as 2+
0.8
0.6
0.4
0.2
0.0
15890
15895
15900
15905
15910
15915
A4V holo in pure water_000001.d: +MS, Deconvoluted (MaxEnt)
A4V holo in pure water_000001.d: C681H1083N203O225S2CuZn, M,15895.76
15920
15925
15930 m/z
Metalloprotein MS Applications:
Effects of Metals on Protein Structure Using Charge State Distribution
Purification of metallated and apo SOD1 (multiple charge states) within the mass spectrometer
Quadrupole
mass filter
to eliminate
low m/z
As
Isolated
0.2 kcal/mol
Time-Of-Flight cutoff
to eliminate high m/z
Metalloprotein MS Applications:
Effects of Metals on Protein Structure Using H/D Exchange
WT (left panel) versus fALS mutant G85R (right panel)
superoxide dismutase showing percentage deuteration levels.
Molnar K S et al. J. Biol. Chem. 2009;284:30965-30973
H/D Exchange: 13 fALS SOD1 Variants
Intact Protein H/D‐Ex MS: Cu and Zn loss
220
200
G41S
Cu+Zn
Mixture
160
Δ mass (Da)
apo
180
140
120
100
80
60
WT
Cu+Zn
G41S (+8) holo
40
WT (+8) holo
20
G41S (+12) apo
0
0.0
20.0
40.0
time (min)
Loss of metal: enormous structural consequence
Apo G41S
Apo G41S
Apo WT
Cu/Zn G41S and WT
Cu/Zn WT
0.2 kcal/mol
Lose Cu/Zn >> structural consequence than mutation
Metalloprotein MS Applications:
Metal Oxidation State and Nuclearity
Figure 8 ESI-FTICR mass spectrum of [3Fe-4S]ferredoxin from Pf (top) in which the intact cluster can be seen at m/z 1866.
Peaks at higher mass-to-charge ratios represent oxygen, sodium, and sulfur adducts, whereas more intense peaks at lower
mass-to-charge ratios represent losses of inorganic sulfide from the cluster. The mass spectrum of 34S labeled [3Fe-434S]
protein from Pf is also shown (bottom). The degradation of the protein is similar to the wild-type [3Fe-4S] protein. The loss of
inorganic sulfide from the iron−sulfur cluster is confirmed by this example, as the mass difference between the sulfur-loss peaks
is 34 Da, whereas in the unlabeled form the difference is 32 Da.
Published in: Keith A. Johnson; Marc F. J. M. Verhagen; Phillip S. Brereton; Michael W. W. Adams; I. Jonathan Amster; Anal. Chem. 2000, 72,
1410-1418.
DOI: 10.1021/ac991183e
Copyright © 2000 American Chemical Society