Post on 15-Apr-2022
proteins
peptides
Mass Spectrometry
Rick Edmondson Ph.D.
Associate Professor of Medicine
Myeloma Institute for Research and TherapyRDEdmondson@uams.edu
• Basics of a mass spectrometer
• Ionization methods
– MALDI
– Electrospray
• Types of mass spectrometers
– TOF
– Ion Trap
– FTICR/Orbitrap
• Carbon isotopes & mass measurement
Mass Spectrometry
“A very big thing that weighs very small things”
Inlet
How does the sample
get Into the instrument
Ionization Source
Solid or liquid sample
has to get into the gas phase
Mass Analyzer
Vacuum Chamber that somehow separates molecules
that differ in mass
Detector
Convert ions into an electronic signal that can be measured
Data System
Computer software and hardware that integrates all the above
Forming gas phase ions is the first step
Mass spectrometers do not measure mass...
They measure mass-to-charge (m/z)
Two ionization methods relevant to Proteomics
MALDI
Matrix Assisted Laser Desorption Ionization
ESI
Electrospray (Nanospray – nanoliter/min flow rates)
MALDI Matrices
Electrospray (ESI)
University of Bristol
http://www.bris.ac.uk/nerclsmsf/techniques/hplcms.html
Electrospray
Easily coupled to HPLC separation
Electrospray
Solvent Evaporation
Coulombic Explosion
Winkler R.: ESIprot: A universal tool for charge state determination and molecular weight calculation of proteins from
electrospray ionization mass spectrometry data. Rapid Commun Mass Spectrom, 24(3), 285-294, 2010,
http://dx.doi.org/10.1002/rcm.4384
ESI leads to multiple charges per molecule
(n +1)
n
y
x
n = (x-1)/(y-x)
http://www.giga.ulg.ac.be
Time of Flight
Ion Trap
http://www.iontrap.umd.edu/research/fast/2DIonTrapAnimation.gif
Hybrid Instruments/ Orbitrap
Unified Atomic Mass Unit (amu) (u)= Dalton (Da)
Thompson (Th) = Mass to charge ratio
Mass Spectrometry Mass Vocabulary
High Resolution Mass Spectrometry
(and Einstein’s Theory of Relativity)
“If you can't explain it to a six year old, you don't understand it yourself.”
― Albert Einstein
Mass Deficiency and Mass Sufficiency
Nominal Mass is close but not the same as isotopic mass
12C, by definition is 12.000000
The Unified atomic mass unit (u) is defined as 1/12 the mass of 12C
1.66055x10-27 kg
1.007 276 467 u Proton
1.008 664 916 u Neutron
0.000 544 662 u Electron
When 1 + 1 does not = 2
Take Oxygen as an example
8 protons + 8 neutrons + 8 electrons =
16.13189
0xygen mass is 15.99491
∆∆∆∆=0.13698
When 1+1≠2…..Use E=MC2 to bail yourself out
Mass Deficiency
The mass of an isotope is less than the nominal mass
Mass Sufficiency
The mass of an isotope is more than the nominal mass
(only H, He, Li, Be, B, N)
Mass defect, mass excess and fractional mass: the driving principle behind
accurate mass-based decisions is the concept of nuclear mass defect,
defined as the difference between the sum of the masses of the constituent
nucleons and the measured exact mass of an atom (Inczedy, 1998). This
difference is an instance of Einstein's special theory of relativity stating that
mass and energy are interchangeable (Einstein, 1905): the nuclear mass
defect accounts for the nuclear binding energy and is always non-negative.
The concept of mass excess is a direct consequence of the nuclear mass
defect: it is defined as the difference d=mobs−mnom between the observed
mass mobs and the nominal mass mnom of an element or compound. In
particular, the mass excess of 12C is defined as zero and mass excesses of
other elements can either be positive (e.g. 1H: 1.00783 and 14N: 14.00307)
or negative (e.g. 16O: 15.99491, 32S: 31.97207, 31P: 30.97376 and 127I:
126.90447)
Kirchner M et al. Bioinformatics 2010;26:791-797
A good written explanation of the nuclear mass defect
Mass excess plots for the human protein phosphorylation sequence database training datasets with theoretical modifications.
Kirchner M et al. Bioinformatics 2010;26:791-797
P 30.973 7615
O 15.994 9146
Why worry about the minutia?
Mass Measurement Accuracy on modern mass spectrometers is truly amazing
routinely better than 1 part per million (ppm)
Use accurate monoisotopic mass values and as many decimals as you can (at
least 5 after the decimal) when calculating modification masses.