Mass Spectrometry I Basic Data Processing. Mass spectrometry A mass spectrometer measures molecular masses. The mass unit is called dalton, which is 1/12.

Mass Spectrometry I

Basic Data Processing

Mass spectrometry

• A mass spectrometer measures molecular masses.

• The mass unit is called dalton, which is 1/12 of the mass of a carbon atom, and is about the mass of one hydrogen atom.

• If there is a mixture of different molecules in a sample, all the masses are measured simultaneously. So you get a spectrum.

Some PicturesMALDI-R Q-Tof Micro

FT-ICR LTQ-Orbitrap

Each peak corresponds to a different type of molecule in your sample

1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000m/z0

100

%

2790.22

1324.60

1265.62

1179.41

2789.22

1325.62

2466.18

2465.20

1759.931326.60

1477.62

1327.611460.59

1748.86

1478.611540.63

1974.94

1760.93

1761.92

1975.93

2356.102355.111976.92

2179.87

2467.19

2468.20

2469.172746.23

2791.23

2792.23

2793.23

3104.412794.20

3103.432795.06 3106.42

...…2789.22 3597.02790.22 5018.02791.23 4406.02792.23 2868.02793.23 1234.0……

peak list

Three Components of an MS• A typical mass spectrometer contains

– Ionizer– Mass analyzer– Detector

• Ion source charges the to-be-measured molecules.– Charge can be negative but often positive.– Two common types: MALDI and ESI.– John B. Fenn & Koichi Tanaka 2002 Nobel Prize in Chemistry

for Electrospray and MALDI

• Mass analyzer separates ions according to the mass to charge ratio (m/z) of the ions.– Iontrap, TOF, Quadrupole, FTICR.

• Detector detects the ions.

Matrix Assisted Laser Desorption/Ionization

Formation of singly charged ions

Sample is co-crystallized with matrix (solid)

Koichi Tanaka, Nobel Prize 2002

Ionization (1): MALDI

Other ionization method exists.

Mass Analyzer (1) – TOF

• Time of Flight.

+ -

+

Detector

Time of flight is proportional to sqrt(m/z)

Other mass analyzer exists.

Drift region (D)

MALDI Time-of-flight

Putting Them Together

MALDI TOF

Average time in TOF: 10-7 sec : average speed 1-2 x 105 km/h

MALDI-TOF Linear

Mass range = 800-200,000

Sensitivity and accuracy decrease rapidly with size !

MALDI-TOF Linear vs Reflectron Mode

Reflectron gives much better resolution for mass < 6,000

• Linear = poor resolution due to velocity variation of ions with the same m/z •Reflectron = Contact lens for a near sighted machine!

Protein “identification” with intact mass

• We measure the intact mass of the protein.• Then search in the protein database to find a

protein with the same mass.• Good idea but there are too many proteins

with the same mass.• In the rest of the lecture we study more

sophisticated methods and why protein ID is important.

Complications

isotopes

widened peaks

profile

Centroiding

Another example with lower resolution

Isotopes

Chemical Composition of Living Matter27 of 92 natural elements are essential.  Elements in biomolecules (organic matter): H, C, N, O, P, S These elements represent approximately 92% of

dry weight.

Organic Matter Organized in "building blocks"

amino acids polypeptides ( proteins)

monosaccharides starch, glycogen

nucleic acids DNA, RNA 

Back to Basics…

element nominal exact Percent average

mass mass abundance mass

 C 12 12.00000 98.9%

13 13.00335 1.1% 12.00115

H 1 1.00783 99.98%

2 2.0140 0.02% 1.008665

O 16 15.99491 99.8% 18 17.9992 0.02% 15.994

 N 14 14.00307 99.63%

15 15.00011 0.37% 14.0067

S 32 31.97207 94.93%

33 32.97146 0.76%

34 33.96787 4.29% excercise

Mass (Weights) of Atoms and Molecules

 Ethyl acetate C4H8O2

4 C12 4 x 12.0000 48.0000 8 H1 8 x 1.0078 8.064 2 O16 2 x 15.99949 31.9898

Nominal Mass: 48 + 8 + 32 = 88

Monoisotopic Mass: 88.0555

Average Mass: 48.04446 + 8.06932 + 31.988 = 88.10178  

Mass or Molecular Weight of molecules

Amino Acids• There are 20 amino acids. All have the

same basic structure but with different side chains:

• Examples: side chain group

H

Glycine, or Gly, or G

Arginine, or Arg, or R

All the 20 Structures

* Picture copied from Dr. R.J. Huskey’s website: http://www.people.virginia.edu/~rjh9u/aminacid.html

Peptides and Proteins

H

Glycine, or Gly, or G

Arginine, or Arg, or R

GR

peptide bonds

N-terminal C-terminal

Exact Mass of Amino Acid Residues in Proteins

Gly G 57.02150Ala A 71.03720Gln Q 128.05860Lys K 128.09500Glu E 129.04270

Note: Leu (L) = Ile (I) = 113.08410

Mass of Amino Acids Residues

Amino Acid Table

AA Codes Mono.

IONSOURCE.COM

AA Codes Mono.Gly G 57.021464 Asp D 115.02694Ala A 71.037114 Gln Q 128.05858Ser S 87.032029 Lys K 128.09496Pro P 97.052764 Glu E 129.04259Val V 99.068414 Met M 131.04048Thr T 101.04768 His H 137.05891Cys C 103.00919 Phe F 147.06841Leu L 113.08406 Arg R 156.10111Ile I 113.08406 CMC 161.01467

Asn N 114.04293 Tyr Y 163.06333

- - - Trp W 186.07931

Cysteine

Proteins are often treated so that cysteine becomes carboxyamidomethyl cysteine (CamC) or Carboxymethyl (CmC) in order to break the disulphide bonds.CamC = 160.03

tripeptide (MW 71.04+87.03+147.07+18.01)=323.15More precisely: monoisotopic mass 323.1481 average mass 323.3490

Ala-Ser-Phe (ASF)

Mass of Peptides and Proteins

In a mass spectrum

323.15 324.15 325.15

Deconvolution adds all the isotopic peaks to the monoisotopic peak. So, the later process does not need to worry about the isotopes.Monoisotope peak

isotope peaks

Check the difference

ESI and Multiply Charged Ions

Electrospray

Electrospray Ionization: Formation of Charged Droplets

Formation of multiply charged ions

Ionization (2) – ESI

Multiply Charged Ions• The same molecules may be charged

differently, and therefore form a few peaks in the spectrum.

323.15

324.15

325.15

162.08

162.58

163.08

m/z(M+2)/2(M+3)/3

For protein/peptide with positive charges, the charge is obtained from adding protons (which has mass approx. 1 dalton. As a result, a molecule with mass M will have peaks at (M+Z)/Z

(M+1)/1

How to determine charge states?

• Isotope ions when resolution is enough.• Check different charge states when resolution

is not enough.

Exercise

395.73

396.22

397.24

Exercise

Exercise

(A) “Multi-charge envelope” (B) After “Charge-deconvolution algorithm”

1541.9

1413.21304.7

1211.9

Baseline

Baseline correction

Convex Hull Method

convex

not convex

Convex Hull

• A convex hull is such that all the data points are above the lines and their extensions.

How to calculate convex hull?

• Stack S contains all the data points that form the convex hull so far.• Data point D[i] = (D[i].x, D[i].y).

Algorithm:

1. S.push( D[0] ); s.push(D[1])2. for i from 2 to n2.1 while D[i], S.top(), S.secondtop() are concave2.1.1 S.pop();2.2 S.push(D[i]);3. return S

S.top()

S.secondtop() D[i]

Analyze the convex hull algorithm

• Correctness– The algorithm finishes.– The output is a convex hull.– The proof will be included in an assignment.

• Time complexity– O(n) time.– Proof: each point is checked only once, and added

to (and therefore removed from) the stack at most once.

Summarize of spectrum preprocessing

• Baseline correction• Centroiding• Charge recognition and deconvolution• Noise removal