Mass Spectrometry MALDI-TOF ESI/MS/MS Mass spectrometer Basic - - PDF document

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Mass Spectrometry MALDI-TOF ESI/MS/MS Mass spectrometer Basic - - PDF document

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11/29/2012 Mass Spectrometry MALDI-TOF ESI/MS/MS Mass spectrometer Basic components Ionization source Mass analyzer Detector 1 11/29/2012 Principles of Mass Spectrometry Proteins are separated by mass to charge ratio

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SLIDE 1

11/29/2012 1

Mass Spectrometry

MALDI-TOF ESI/MS/MS

Mass spectrometer

  • Basic components

– Ionization source – Mass analyzer – Detector

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SLIDE 2

11/29/2012 2

Principles of Mass Spectrometry

  • Proteins are separated by mass to

charge ratio (limit 1 charge/1.5-2kDa)

  • Charge occurs through ionization
  • Most common ionization methods in

proteomics

– Matrix assisted laser desorption ionization (MALDI) – Electro-spray ionization (ESI)

Electro-Spray Ionization

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SLIDE 3

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ESI

  • Advantages

– Samples are in solution – Small sample volumes and sizes (l/min) – Can be coupled to HPLC

  • (nano-HPLC or UHPLC)

– Can be run in both positive and negative mode – Results in multiple charging so larger proteins can be measured

  • Disadvantages

– Not all molecules will ionize – High maintenance – Only uses small fraction of the sample

Multiple charging of Proteins

200 1000 2000

m/z +7 1766.6 +8 1545.7 +9 1374.2 +10 1236.9 +11 1124.6 +12 1031.0 +13 951.8 +14 884.0 +15 825.0 +16

772.4

+17 727.5

50 100 Relative Abundance

Cytochrome C

Charge size est MW 17 727.50 12367.50 16 772.40 12358.40 15 825.00 12375.00 14 884.00 12376.00 13 951.80 12373.40 12 1031.00 12372.00 11 1124.60 12370.60 10 1236.90 12369.00 9 1374.20 12367.80 8 1545.70 12365.60 7 1766.60 12366.20 Avg 12369.23 Stdev 4.77

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Deconvoluted Data

10000

mass

12500 15000

50 100 Relative Abundance

12369 +/- 2

Determining Ion Charge

  • Charge is calculated from the separation
  • f the peaks in a resolved isotope series.

– MALDI gives singly charge ions (usually) – ESI gives multiply charged ions

SINGLY CHARGED  1 DOUBLY CHARGED  0.5

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Matrix Assisted Laser Desorption Ionization

  • Samples are mixed with a matrix and

placed on the surface of a target

  • Target is placed inside the vacuum of MS
  • Samples are ionized by high energy laser
  • Most/all samples ionize
  • Usually single charge

MALDI

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SLIDE 6

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Mass Analyzers

  • Quadrapole
  • Time of Flight (TOF)
  • Ion Trap
  • Fourier Transformed Ion Cyclotron
  • 2000V

+

Mass Spectrometry Basics

+2 +2 +1 +1

+ pole

  • pole

Heavy ions Light Ions

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SLIDE 7

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Quadrapole

http://hk.youtube.com/watch?v=8AQaFdI1Yow&NR=1 http://www.chemistry.adelaide.edu.au/external/soc-rel/content/quadrupo.htm http://www.youtube.com/watch?feature=endscreen&v=WbX27Gg5ziU&NR=1

Time of Flight

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Ion Trap

Nature Reviews Drug Discovery 2, 140-150 (February 2003) http://www.youtube.com/watch?v=KjUQYuy3msA&feature=related http://www.youtube.com/watch?v=3uUwa1DDoHQ

Fourier transformed ion cyclotron resonance

http://hk.youtube.com/watch?v=a5aLlm9q-Xc&feature=related www.pnl.gov/news/release.asp?id=249

FTICR_WMKeck_NCSU video of FTICR and how it works no sound

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SLIDE 9

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Using MS Data

  • So how do we use these?

– Full mass – Mass of complexes – Peptide map – Sequencing for identification – Quantitation

MALDI-TOF Peptide Map

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Protein Sequencing

  • Process

– Protein digested with protease

  • Typically trypsin which cleaves at K and R

– Peptides separated by HPLC (nano-HPLC) – Analyzed by MS/MS

  • Several problems exist

– De novo sequencing is very difficult – Fragments may be too large or not sufficiently charged – Poor ionization of fragments – Post translational modifications

MS sequencing

  • 1. Sample is injected into

reverse phase HPLC and peptides separated.

  • 2. Fragments are separated

by mass in first quadrapole mass analyzer

  • 3. Selected ions enter

second quadrapole analyzer and mixed with argon to fragment peptides.

  • 4. Daughter ions are

analyzed by TOF mass spectrometer.

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Fragmentation of Peptides

http://www.matrixscience.com/help/fragmentation_help.html

Peptide Sequence

100 250 500 750 1000 m/z % Intensity K 1166 L 1020 E 907 D 778 E 663 E 534 L 405 F 292 G 145 S 88 b ions 147 260 389 504 633 762 875 1022 1080 1166 y ions

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SLIDE 12

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Amino Acid Masses

Amino acid Mass(avg) Amino acid Mass(avg) G 57.0520 D 115.0886 A 71.0788 Q 128.1308 S 87.0782 K 128.1742 P 97.1167 E 129.1155 V 99.1326 M 131.1986 T 101.1051 H 137.1412 C 103.1448 F 147.1766 I 113.1595 R 156.1876 L 113.1595 Y 163.1760 N 114.1039 W 186.2133

Ambiguous Masses

Amino acid combination Mass (amu) Single amino acid Acetylated amino acid Mass (amu) Unmodified amino acid

G-G 114.104 N

114.1039

Ac-G 99.09 V

99.1236

G-A 128.1308 K/Q

128.1308 128.1742

Ac-A 113.1225 L/I

113.1595

V-G 156.1378 R

156.1876

Ac-S 129.1219 E

129.1155

G-E 186.1675 W

186.2133

Ac-N 156.1509 R

156.1876

A-D 186.1674 W

186.2133

S-V 186.2108 W

186.2133

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Closely Related Sequences

ATSARA1A MFLFDWFYGI LASLGLCKKE AKILFLGLDN AGKTTLLHML ATSARA1B MFLFDWFYGI LASLGLWQKE AKILFLGLDN AGKTTLLHML ATSARA1A KDERLVQHQP TQHPTSEELS IGKINFKAFD LGGHQIARRV ATSARA1B KDERLVQHQP TQHPTSEELS IGKIKFKAFD LGGHQIARRV ATSARA1A WKDCYAKVDA VVYLVDAYDR DRFVESKREL DALLSDEALA ATSARA1B WKDYYAKVDA VVYLVDAYDK ERFAESKREL DALLSDEALA ATSARA1A

NVPCLILGNK IDIPYASSED ELRYYLGLTN FTTGKGIVNL

ATSARA1B

TVPFLILGNK IDIPYAASED ELRYHLGLTN FTTGKGKVTL

ATSARA1A

EDSGVRPLEV FMCSIVRKMG YGEGFKWLSQ YIK

ATSARA1B

GDSGVRPLEV FMCSIVRKMG YGEGFKWLSQ YIN

ATSARA1A pI = 6.10 MW = 21952.33 ATSARA1b pI = 6.52 MW = 21972.45

ATSAR1B ATSAR1A Mass AA Sequence Mass AA Sequence 2335.198 1-19

MFLFDWFYGILASLGLWQK

2124.07 1-18

MFLFDWFYGILASLGLCK

292.3475 20-22

EAK

292.348 20-22

EAK

1160.667 23-33

ILFLGLDNAGK

1160.667 23-33

ILFLGLDNAGK

956.5597 34-41

TTLLHMLK

956.5597 34-41

TTLLHMLK

2129.099 45-63

LVQHQPTQHPTSEELSIGK

2129.099 45-63

LVQHQPTQHPTSEELSIGK

233.3232 64-65

IK

521.3082 64-67

INFK

257.3403 66-67

FK

1184.617 68-78

AFDLGGHQIAR

1184.617 68-78

AFDLGGHQIAR

659.3035 83-87

DYYAK

599.2494 83-87

DCYAK

1469.752 88-100

VDAVVYLVDAYDK

1497.758 88-100

VDAVVYLVDAYDR

581.2929 103-107

FAESK

609.3242 103-107

FVESK

2342.285 109-130 ELDALLSDEALATVPFLILGN

K

2311.221 109-130 ELDALLSDEALANVPCLILG

NK

1491.733 131-143

IDIPYAASEDELR

1507.727 131-143

IDIPYASSEDELR

1351.7 144-155

YHLGLTNFTTGK

1377.705 144-155

YYLGLTNFTTGK

2178.141 158-177

VTLGDSGVRPLEVFMCSIVR

2433.263 156-177 GIVNLEDSGVRPLEVFMCSI

VR

888.392 179-186

MGYGEGFK

888.392 179-186

MGYGEGFK

923.4621 187-193

WLSQYIN

937.5142 187-193

WLSQYIK

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11/29/2012 14

How do we deal with this?

  • Use available information

– The genome – Edman sequences – Comparison to known proteins

  • Use programs such as Protein Prophet,

Sequest, Mascot, etc.

Sequencing with MS/MS

  • Currently three main search engine

programs are used to identify sequences rather than creating the sequence from the data.

– SEQUEST (Xcor values > 1.9, 2.2, or 3.7 for ions of 1, 2, or 3 charges are usually accurate) – Mascot (Scores of >40-50 give good assignments) – X!Tandem (hyperscore, the larger the better)

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Sequencing with MS/MS

  • This process requires that the peptide

be from a protein that the sequence is known.

– From an organism with a sequenced and anotated genome. – Protein was purified and sequenced. – Present in an EST library. – Has identity or high similarity with a protein from another organism.

Quantification by MS

  • SILAC (stable isotope labeling of

amino acids in cell culture)

–In vivo labeling with C13 or N15

  • ICAT (Isotope coded affinity tag)
  • iTRAQ (Isobaric tag for relative and

absolute quantitation)

  • Competing technology

–DIGE (Differential Gel Electrophoresis)

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SILAC ICAT-label

  • 4 parts to ICAT molecule

–A protein reactive group – Iodoacetamide

  • covalently links to free cysteines.

–An affinity tag – biotin

  • concentrates the cysteine-containing

peptides, reducing complexity.

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ICAT-label

  • An isotopically labeled linker

(C10H17N3O3)

– The linker chain can substitute up to nine 13C atoms. – The light and heavy molecules are chemically identical – Comparison of labeled peptides provides a ratio of the protein concentration in the

  • riginal sample.
  • http://www.bio.davidson.edu/courses/GENOMICS/ICAT/ICAT.swf

ICAT-label

  • An acid cleavage site:

– remove biotin and part of the linker by adding TFA. – reduces the mass of the tag – improves the overall peptide fragmentation efficiency.

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iTRAQ

  • Label up to 8 samples at once
  • Amine specific labeling (lysine and N-

terminal) (N-hydroxysuccinamide)

  • Mass of all labels the same.

– The tag consists of the reactive group, a reporter molecule and a linker to balance the masses. – During fragmentation in MS the reporter group is released.

  • After fragmentation reporter labels are

found between m/z 113 and m/z 121

  • Ratio of peaks of reporter ions is

proportional to relative concentrations.

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Sensitivity of Different Methods

  • Silver stain

below 1 ng (linear 1-60 ng)

  • Colloidal coomassie blue 100 ng/protein spot
  • Deep Purple

<1 ng

  • Sypro ruby

1 ng (linear 1-1000 ng)

  • DIGE

0.125 ng (linear 0.125 ng-10 g)

  • MS

500 fM

  • FTICR MS

500 aM – For a 60 kDa protein 500 fM = 30 ng 500 aM = 30 pg