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.