Mass spectrometry in proteomics

1
Mass spectrometry in proteomics
2
The goals of proteomics

• Proteomics aims to simultaneously characterize
  all the proteins in biological samples
• Identify / sequence the proteins and determine
  their relative abundances
• Characterize their posttranslational
  modifications
• Determine the three dimensional structure
• Identify the interactions between the proteins,
  and with other molecules most proteins function
  in complexes
• Follow all the above during development, in
  response to hormonal stimuli, in health and
  disease, etc.

3
More goals for proteomics

• Sub-cellular location, organelles and membranes
• Turnover kinetics, degradation and synthesis
  rates
• Biomarkers discovery, early detection, targets
  for drugs and immunotherapy, personalized and
  bedside proteomics
• Differences between cells, requires proteomics
  analyses of single cells or small number of cells
• Using mass spectrometry as microscopy
• Deciphering signaling pathways

4
NanoSpray Tip types
5
NanoSpray Flow Rates
6
Perfect NanoSpray
7
NanoSpray Tips
8
Identification by MALDI
9
350 min grad on 60 cm column (sample
Francesca Forner, viewer Jorgen Cox, Mann lab)
10
LCQ, electrospray quadrupole ion trap mass
spectrometer
11
ESI-Q-Tof
12
Identification by MS/MS
13
ETD Concept Ion / Ion Chemistry
M 3H2
C2H1 ZH1
C
Z

• Made by electron capture
• ionization in a chemical
• ionization source

14
Ion-ion Reactions for ETD
Front
Center
Back
Step 4 RF lens off and ready to scan
15
LTQ XL with ETD Allows Alternating CID and ETD
Scans
CID fragmentation
y
y
y
z
z
z
c
c
b
b
b
c
ETD fragmentation
16
LTQ XL with ETD Instrument Configuration

DSQ EI/CI Source
Coarse Quadrupole Mass Filter (Shares Q00/Q1 DC
RF)
Lens 1,2,3,4 L2Gate
Detector 1
Filament
e-
Center
Changeable Ion Volume
Section
Transfer Line Heater Zone
Heated Transfer Line

Front
Back
Detector 2
Lens
Lens
Fused Silica
Heated Dual Restrictor Enclosure
Back
Front
Section
Section
Reagent 1 Heated Inlet
Reagent 2 Heated Inlet
N2 CI/Carrier Gas
17
Freedom of Dissociation!

• CID
• MSA
• PQD
• ETD
• HCD
• ECD
• IRMPD
• Because not all peptides are created equal!

18
Protein Concentrations HUPO PPP Specimen BDAA01
C g/L
Analytes
19
Dynamic range (serum, CNBr, MSight)
20
Mass spectrometers as microscope
21
Mass spectral imaging method (Caprioli,
Vanderbilt)
22
Mass spectral imaging results (Caprioli,
Vanderbilt)
23
Protein Concentrations HUPO PPP Specimen BDAA01
C g/L
Analytes
24
Plasma proteins
(top ten abundant proteins removalgt only
10-fold increased sensitivity)
25
Protein complexes (Sharon and Robinson 07)
26
Protein complexes 2
27
Protein complexes (Sharon and Robinson 07)c
28
PTM phosphorylation

• Phosphate groups are attached to S,T and Y mostly
  and sometimes to D, H.
• The phosphate falls off easily at high pH and due
  to activity of phosphataes.
• Beta elimination and dehydro alanine formation
• Loss of phosphate from S and T in the source,
  trap or during MS/MS.
• Pi-Tyr is very rare.
• Enrichment is needed. Ion exchange, straight
  silica, metal affinity binding.
• Loss of 80, 98 or 49

29
Phosphorylation
Phospho-peptides are detected with the added mass
of 80Da (PO3). However, when they are fragmented
(MS/MS) 98Da are lost (H2PO4)
MH - H3PO4 doubly charged
30
PTM glycosylation
31
Ubiquitination
Ub-protein XR/XXXXKXXXK/X ubiquitin
R/GG (114Da)
Ubiquitinated Lys
32
Intracllular localization

• Disruption and separation of memebranes from
  soluble proteins
• Separation of nuclei from mitochondria
• Detergent extraction of membrane
• Mass spectrometry compatible detergents need to
  be used.

33
Protein Correlation Profiling(Foster and Mann 06)
34
Mass segmentation