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Mass Spectrometry Overview and Mass Spectrometry of Proteins and Glycoproteins. David Graham, Ph.D. Assistant Professor, Department of Molecular and Comparative Pathobiology School of Medicine Director for The Center for Resources in Integrative Biology dgraham@jhmi.edu. Goals .

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mass spectrometry overview and mass spectrometry of proteins and glycoproteins

Mass Spectrometry Overview and Mass Spectrometry of Proteins and Glycoproteins

David Graham, Ph.D.

Assistant Professor,

Department of Molecular and Comparative Pathobiology

School of Medicine Director for

The Center for Resources in Integrative Biology

dgraham@jhmi.edu

goals
Goals
  • Better Understanding of Mass Spectrometry
    • Basic introduction
    • Components of MS
    • Basic Principles
    • Types of instruments
    • MS as applied to proteins, peptides and glycopeptides
    • ECD/ETD
  • Analyzing MS data
    • Software tools
    • Workflows
    • Extracting Biological Meaning
sources
Sources:
  • Agard lab
    • www.msg.ucsf.edu/agard/maldi/IntrotoMS.ppt
  • Cobb lab
    • shadow.eas.gatech.edu/~kcobb/isochem/lectures/lecture2_massspec.ppt‎
  • ME 330.804: Mass Spectrometry in an “Omics” World
    • Johns Hopkins – multiple faculty contributers
  • MAMSLAB: Slides from the late Robert Cotter
  • Books:
  • Mass Spectrometry of Glycoproteins : Methods and Protocols
  • Editor(s): Jennifer J. Kohler1, Steven M. Patrie2
  • Mass Spectrometry of Proteins and Peptides : Mass Spectrometry of Proteins and Peptides
  • Editor(s): John R. Chapman1
    • Both available through welch medical library online
slide4
More..
  • The Expanding Role of Mass Spectrometry in Biotechnology,GarySiuzdak (2nd edition 2006) ISBN 0-9742451-0-0
  • Mass Spectrometry Desk Reference, O. David Sparkman (2000, 1st edition) ISBN 0-9660813-2-3
  • MassSpectrometry of BiologicalMaterials, Barbara S. Larsen& Charles N. McEwen (2nd. Edition 1998) ISBN 978-0824701574
  • ProteinsandProteomics: A LaboratoryManual, editedby Richard Simpson (2003) ISBN 0-87969-554-4
  • MassSpectrometryin Biophysics: Conformationand Dynamics of Biomolecules, Igor A. KaltashovandStephen J. Eyles (2005) ISBN 0-471-45602-0
  • Time-of-Flight MassSpectrometry: Instrumentation andApplications in BiologicalResearch, Robert J. Cotter (1997) ISBN 0-8412-3474-4
  • Disclaimer– besteffort has beenmadetoreferenceoriginalsources. Pleasecontactdgraham@jhmi.eduforcorrection of anyerrorsorommisions.
mass spectrometry is applied physics
Mass spectrometry is applied physics
  • Magnetism
  • Newtons laws of motion
  • Basic tennants are dealing with charged molecules
  • Two laws:
    • Lorenz force law:
    • If a particle of charge q moves with velocity v in the presence of an electric field E and a magnetic field B, then it will experience a force (F)
    • Newtons second law (non-relatavistic motion):
      • F=ma
    • The terms F can be related and the equation derived:
      • (m/q)a= E + v x B
basic equations governing mass spectrometry
Basic equations governing mass spectrometry

Ion’s kinetic E function of accelerating voltage (V) and charge (z).

Centrifugal force

Applied magnetic field

balance as ion goes through flight tube

Combine equations to obtain:

Fundamental equation of mass spectrometry

Change ‘mass-to-charge’ (m/z) ratio by

changing V or changing B.

NOTE: if B, V, z constant, then:

Cobb lab

what is the take home point
What is the take home point?
  • We can control our voltages
  • We know our distances
  • We know our field strengths
  • Thus:
    • A simple set of equations can be used to calculate the m/z for all different types of mass spectrometers
basic components of a mass spectrometer

Ion source:makes ions

Basic components of a mass spectrometer

Sample

Mass analyzer: separates ions

Detector:presents information

Modified from Agard lab

slide9

Mass Spectrometer Block Diagram

High Vacuum System

Ion

source

Mass

Analyzer

Data

System

Inlet

Detector

Modified from Agard lab

slide10

Mass Spectrometer Block Diagram

Turbo pumps

High Vacuum System

Ion

source

Mass

Analyzer

Data

System

Inlet

Detector

Modified from Agard lab

slide11

Sample Introduction

High Vacuum System

Ion

Source

Mass

Analyzer

Data

System

Inlet

Detector

HPLC

Flow injection

Sample plate

Modified from Agard lab

slide12

Ion Source

High Vacuum System

Ion

Source

Mass

Analyzer

Data

System

Inlet

Detector

MALDI

ESI

FAB

SIMS

EI

CI

Modified from Agard lab

slide13

Ion Sources make ions from sample molecules(Ionization is required to move and detect molecules.)

Partialvacuum

Sample Inlet Nozzle

(Lower Voltage)

Pressure = 1 atmInner tube diam. = 100 um

MH+

N2

+

+

+

+

+

+

+

+

+

+

+

+

MH2+

+

+

+

+

+

+

+

+

+

+

+

+

Sample in solution

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

N2 gas

+

+

+

+

+

MH3+

High voltage applied

to metal sheath (~4 kV)

Charged droplets

Electrospray ionization:

Introduced by John Fenn (Nobel Prize 2002):

Yamashita, M.; Fenn, J.B., J. Phys. Chem. 88 (1984) 4451.

Whitehouse, C.M.; Dreyer, R.N.; Yamashita, M.; Fenn, J.B., Anal. Chem. 57 (1985) 675.

Fenn, J.B.; Mann, M.; Meng, C.K.; Wong, S.F.; Whitehouse, C.M., Science 246 (1989) 64.

Sources: Agard lab and MAMSLAB

slide14

Favors ejection of multiply charged

Ions

Based on an ion evaporation model:

Iribarne, J.V.; Thomson, B.A., J. Chem. Phys. 64 (1976) 2287.

Thomson, B.A.; Iribarne, J.V., J. Chem. Phys. 71 (1979) 4451.

Sources: Agard lab and MAMSLAB

assisted electrospray

Low Voltage (0.5 kv)

High Voltage (5 kv)

Low Voltage (0.1 kv)

MS

LC Column Flow

Drying

Gas

Nebulizing

Gas

Assisted Electrospray

www.e-cats.com/chemistry/01measurements/IntrotoMassSpec.ppt

slide16

MALDI: Matrix Assisted Laser Desorption Ionization

Sample plate

Laser

  • Sample is mixed with matrix (X) and dried on plate.
  • Matrix absorbs UV or IR energy from laser
  • Matrix ionizes and dissociates; undergoes a phase change to supercompressed gas
  • Some analytes are ionized by proton transfer: XH+ + M  MH+ + X.
  • Matrix expands supersonically and ions are entrained in the plume
  • Koichi Tanaka (Nobel Prize 2002)

hn

MH+

Grid (0 V)

+/- 20 kV

Modified from Agard lab and Cotter lab (MAMSLAB)

common maldi matrices
Common MALDI Matrices

Source: MAMSLAB

slide18

Mass Analyzer

High Vacuum System

Ion

source

Mass

Analyzer

Data

System

Inlet

Detector

Time of flight (TOF)

Quadrupole

Ion Trap

Orbitrap

Magnetic Sector

FTMS

Modified from Agard lab

slide19

Mass analyzers

  • Mass analyzers separate ions based on their mass-to-charge ratio (m/z)
  • Operate under high vacuum (keeps ions from bumping into gas molecules)
  • Actually measure mass-to-charge ratio of ions (m/z)
  • Key specifications are resolution, mass measurement accuracy, and sensitivity.
  • Several kinds exist: for bioanalysis, quadrupole,time-of-flight and ion traps are most used.

Modified from Agard lab

slide20

Quadrupole Mass Analyzer

Uses a combination of RF and DC voltages to operate as a mass filter.

  • Has four parallel metal rods.
  • Lets one mass pass through at a time.
  • Can scan through all masses or sit at one fixed mass.

Modified from Agard lab

slide21

m1

m2

m4

m3

m2

m1

m4

m3

mass scanning mode

m1

m2

m2

m2

m2

m2

m4

m3

single mass transmission mode

Quadrupoles have variable ion transmission modes

Modified from Agard lab

slide22

Time-of-flight (TOF) Mass Analyzer

Source

Drift region (flight tube)

+

+

detector

+

+

V

  • Ions are formed in pulses.
  • The drift region is field free.
  • Measures the time for ions to reach the detector.
  • Small ions reach the detector before large ones.

Modified from Agard lab

slide24

Ion Trap Mass Analyzer (Developed in the 20’s)

Top View

Cut away side view

^ Kingdon KH (1923). "A Method for the Neutralization of Electron Space Charge by Positive Ionization at Very Low Gas Pressures”. Physical Review 21 (4): 408. Bibcode:1923PhRv...21..408K. doi:10.1103/PhysRev.21.408.

ion trap design modified by alexander makarov
Ion Trap Design modified by Alexander Makarov
  • Uses a combination of electrostatic attraction (charge) and centripetal forces
  • Image current is detected as ions orbit central electrode (detected on outer electrode)
  • Data is processed in a similar manner to FTICR data (Fourrier Transformed)

Centrifugal force

Makarov A. (2000). "Electrostatic axially harmonic orbital trapping: A high-performance technique of mass analysis". Analytical Chemistry : AC 72 (6): 1156–62. doi:10.1021/ac991131p.

slide30

Detectors

High Vacuum System

Ion

source

Mass

Analyzer

Data

System

Inlet

Detector

Microchannel Plate

Electron Multiplier

Hybrid with photomultiplier

Modified from Agard lab

microchannel plate detector
Microchannel plate detector

primary ion

-

1000V

+

-

e

-

e

L

-

e

-

e

-

100V

D

L >> D

Modified from Agard lab

slide32

Data System

High Vacuum System

Ion

source

Mass

Analyzer

Data

System

Inlet

Detector

Controller software (VENDOR specific)

Modified from Agard lab

slide33

Summary: acquiring a mass spectrum

Ionization

Mass Sorting (filtering)

Detection

Ion Source

Ion

Detector

Mass Analyzer

  • Form ions
  • (charged molecules)

Sort Ions by Mass (m/z)

  • Detect ions

100

75

Inlet

• Solid

• Liquid

• Vapor

50

25

0

1330

1340

1350

Mass Spectrum

Modified from Agard lab

slide34

40000

30000

20000

10000

0

The mass spectrum shows the results

MALDI TOF spectrum of IgG

MH+

Relative Abundance

(M+2H)2+

(M+3H)3+

50000

100000

150000

200000

Mass (m/z)

Modified from Agard lab

slide35

ESI Spectrum of Trypsinogen (MW 23983)

M + 15 H+

1599.8

M + 16 H+

M + 14 H+

1499.9

1714.1

M + 13 H+

1845.9

1411.9

1999.6

2181.6

m/z

Mass-to-charge ratio

Modified from Agard lab

slide36

Atomic Mass Units

  • Despite being called a Dalton after John Dalton in 1803 who suggested 1H, the discovery of naturally occurring isotopes in 1912 eventually lead to one AMU or Dalton (Da) as being based upon using carbon 12, 12C, as a reference
  • One Dalton is defined as 1/12 the mass of a single carbon-12 atom
  • Thus, one 12C atom has a mass of 12.0000 Da.
isotopes

1981.84

1982.84

1983.84

Isotopes
  • We use isotopes to resolve the charge state of peaks since most element has more than one stable isotope

“Monoisotopic mass”

No 13C atoms (all 12C)

One 13C atom

Mass difference

of 1 Da indicates

a singly charged

Peptide

z=2 delta=0.5

z=3 delta=0.333

z=4 delta=0.25

Etc.

Two 13C atoms

Mass spectrum of peptide with 94 C-atoms (19 amino acid residues)

Modified from Agard lab

slide39

4361.45

4360.45

m/z

Isotope pattern for a larger peptide (207 C-atoms)

Modified from Agard lab

mass spectrum of insulin
Mass spectrum of insulin

2 x 13C

13C

12C: 5730.61

Insulin has 257 C-atoms. Above this mass, the monoisotopic peak is too small to be very useful, and the average mass is usually used.

Modified from Agard lab

monoisotopic mass
Monoisotopic mass

When the isotopes are clearly resolved the monoisotopic mass is used as it is the most accurate measurement.

Modified from Agard lab

average mass
Average mass

Average mass corresponds to the centroid of the unresolved peak cluster

When the isotopes are not resolved, the centroid of the envelope corresponds to the weighted average of all the the isotope peaks in the cluster, which is the same as the average or chemical mass.

Modified from Agard lab

slide43

What if the resolution is not so good?

At lower resolution, the mass measured is the average mass.

Better resolution

Poorer resolution

6130

6140

6150

6160

6170

Mass

Modified from Agard lab

slide44

Resolution =18100

8000

6000

Resolution = 14200

Counts

4000

Resolution = 4500

2000

0

2840

2845

2850

2855

Mass (m/z)

Mass accuracy depends on resolution

15 ppm error

24 ppm error

55 ppm error

Modified from Agard lab

how is resolution calculated
How is resolution calculated?
  • Resolution is the ratio of the mass divided by full width at half maximum. Also known as resolving power
  • R = m/Δmwhere
  • Δm= peak width (FWHM definition)
  • Δm= mass difference between two
  • peaks (valley definition)
  • What mass resolution is required to separate m/z 88 and 89?

m/Δm = 88/1 = 88

Modified from Agard lab /

ME 330.804

typically we perform bottom up proteomics approaches
Typically we perform “bottom up” proteomics approaches
  • Proteins are either chemically cleaved or digested with endopeptidases (most commonly trypsin)

ME 330.804

since resulting peptides follow a repeating pattern

yn-i

xn-i

ai

bi

low energy

high energy

Since resulting peptides follow a repeating pattern..

zn-i

vn-i

wn-i

-HN--CH--CO--NH--CH--CO--NH-

Ri

CH-R’

R”

ci

di+1

we can deduce the sequence of the peptide by subtracting one fragment ion from the next
We can deduce the sequence of the peptide by subtracting one fragment ion from the next
  • Can “read” the sequence N->C using the b-ions and C->N using the y ions

Proteome software

examples of tandem and hybrid instruments
Examples of tandem (and hybrid) instruments:

Tandem in time:

  • Ion trap mass spectrometer (ITMS)
  • Fourier transform mass spectrometer (FTMS)
  • Linear ion trap/FTMS (LTQ-FT)

Tandem in space:

  • Triple quadrupoles
  • Quadrupole/time-of-flight (QTOF)
  • Time-of-flight/time-of-flight (TOF/TOF)
  • Ion trap/time-of-flight (trapTOF, Qit/TOF)

ME 330.804

at jhu
At JHU
  • OrbitrapElite and Velos
  • Mass Range m/z 50 - 2,000, m/z 200 - 4,000
  • Resolution 60,000 at m/z 400 at a scan (FWHM) rate of 4 Hz
  • Minimum resolution 15,000
  • Maximum resolution > 240,000 at m/z 400
  • Dynamic Range
  • > 5,000 within a single scan guaranteeing specified mass accuracy
  • MSn, for n = 1 through 10
  • ETD Option
at jhu1
At JHU

ME 330.804

at jhu2
At JHU

ME 330.804

for intact glycopeptides
For intact glycopeptides
  • Higher energy fragmentation can be used for unambigous identification of sites of N-linked glycan utilization
    • Overcomes the problems associated with PNGaseF and deamidation
    • HCD feature on Orbitrap instrumentation (C-TRAP)
  • High Energy CID by MALDI TOF/TOF
    • Uses Argon as a collision gas
velos with etd option allows for hcd or etd
Velos with ETD option allows for HCD or ETD

and for

glycopeptides

ME 330.804

last steps bioinformatics step 1 data extraction
Last steps: Bioinformatics. Step 1. Data extraction

Mancuso et al., Data extraction from proteomics raw data: An evaluation of nine tandem MS tools using a large Orbitrap data set: JPR: 2012

next choose database
Next: Choose Database

Courtesy R. Gundry

database size affects sensitivity
Database size affects sensitivity
  • Large databases:
    • Unrestricted search (e.g. no-enzyme)
    • Large number of entries
  • Algorithms lose sensitivity as search space is increased (more peptides have to be queried)
  • For both Mascot and Sequest, more correct peptide IDs when used IPI (56,000 entries) vs. NR (1.5 million entries)
  • Mascot is more affected than Sequest
    • In large database searches, Mascot will list the peptides in the top 10, but not list them first (when compare to smaller DB)
    • Sequest better able to rank poorer quality peptides, especially when large database used and unconstrained searches done

Kapp, et. al., Proteomics, 5(13),3475-90

Courtesy R. Gundry

making sense of it all
Making sense of it all
  • Highly recommend Scaffold and Scaffold PTM
  • Can export results from Proteome Discoverer
  • Easy view of experimental findings