High Throughput and Large Scale Proteomics Analysis - PowerPoint PPT Presentation

Loading...

PPT – High Throughput and Large Scale Proteomics Analysis PowerPoint presentation | free to download - id: 1fe286-YWFiN



Loading


The Adobe Flash plugin is needed to view this content

Get the plugin now

View by Category
About This Presentation
Title:

High Throughput and Large Scale Proteomics Analysis

Description:

High Throughput and Large Scale Proteomics Analysis. Austin Yang, Ph.D. ... Ribosomes. 10 mM, 1x 107. Kinases. Cyclins. 1 mM, 1x 106. 0.1 mM, 1x 105 ... – PowerPoint PPT presentation

Number of Views:91
Avg rating:3.0/5.0
Slides: 36
Provided by: austi96
Learn more at: http://www.ipam.ucla.edu
Category:

less

Write a Comment
User Comments (0)
Transcript and Presenter's Notes

Title: High Throughput and Large Scale Proteomics Analysis


1
High Throughput and Large Scale Proteomics
Analysis
Austin Yang, Ph.D. Department of Pharmaceutical
Sciences, University of Southern California
2
Overview
  • Shotgun proteomics and ESI mass spectrometry
  • Proteomic data mining
  • and data visualization

3
12,000 proteins
4
(No Transcript)
5
Are We Ready for Mammalian Proteomics ?
Shotgun Proteomics
2-D Gel
Cytoskelatal Proteins mM, 1x 109 copies/cell
Metabolism
0.1 mM, 1x 108
Ribosomes
10 mM, 1x 107
Kinases
1 mM, 1x 106
Cyclins
0.1 mM, 1x 105
Transcription factors 10 nM, 1x 104
Synaptic Markers 0.1 nM, 1x 103
6
Advantages of Proteomics Using LC-MS/MS
  • No pre-selection of biased targets
  • (hypothesis-free, open approach)
  • Protein variants are detected simultaneously
  • Protein isolation and detection are on a small
    scale ( 10 fmol from complex mixtures
    subcellular fractions, whole cells, or tissue)
  • Obtain sequence information of peptides (not
    just masses) and can sequence 4,000 proteins in
    a single experiment

7
Liquid Chromatography Quadrupole Ion Trap Tandem
Mass Spectrometer
8
Electrospray vs Nanospray
9
Splitless Nano-Liquid Chromatography
10
Five Independent Loop Injections
11
10-cycle MudPIT Analysis
12
Multidimensional Protein Identification
Technology (MudPIT)
Digested protein complexes
20,000 MS/MS spectra/day
13
Isotope-Coded Affinity Tags (ICAT)
14
Electrospray Ionization (ESI)
Ions in gaseous phase
Ions in solution
LC
Spray tip
Ion source opening for the MS
15
(No Transcript)
16
Theoretical CID of a Tryptic Peptide




F
L
G
K


F
L
G
K
b3
y1
F
L
G
K




Parent ions
F
L
G
K
F
L
G
K
CID
b2
y2

F
L
G
K




F
L
G
K
F
L
G
K
b1
y3
Non-dissociated Parent ions
Daughter ions
Relative Intensity
m/z
(464.29)
17
SequestQueue (6,000 dta x50 300,000 ms/ms scans)
18
Data Mining through SEQUEST and PAULA
  • Database Search Time
  • Yeast ORFs (6,351 entries) 52
    sec 0.104 sec/s
  • Non-redundant protein (100k entries) 3500 min
  • EST (100K entries, 3-frames)
    5-10,000 min

19
SEQUEST Algorithm
Theoretical MS/MS spectra
Step 1. Determine Parent Ion molecular mass
Step 2.
500 peptides with masses closest to that of the
parent ion are retrieved from a protein database.
Computer generates a theoretical MS/MS Spectrum
for each peptide sequence (SEQ1, 2, 3, 4, )
(Experimental MS/MS Spectrum)
ZSA-charge assignment
Step 4. Scores are ranked and Protein
Identifications are made based on these cross
correlation scores.
Step 3. Experimental Spectrum is compared with
each theoretical spectra and correlation scores
are assigned.
(Experimental MS/MS Spectrum)
Unified Scoring Function
20
One spectrum TWO protein identifications
Spectrum A was used to search against NCBI human
database Macrophage inhibitory factor was
identified
Same spectrum was used to search
against non-redundant database. Bovine
G-protein gamma was identified. Since the
primary amino acid sequence of human G-protein
gamma is almost identical to bovine, this
protein was later identified as human
G-protein Gamma. The initial false ID was due
to an entry missing of human g-protein in
human database. The sequence was later
reentered Into the human database and the third
search yielded correct ID.
Mol Cell Proteomics. 2003 Jul2(7)428-42.
Fragment ions match both sequences are indicated
by Spectrum B has two additional ions matched
to G-protein gamma
21
Distribution of Xcorr from correctly and
incorrectly identified peptides
22
X-correlation vs Peptide length
23
Distribution of Xcorr vs Charge State
24
F-score and probability-based peptide assignment
25
(No Transcript)
26
(No Transcript)
27
Identification of modified LRP in APP/PS1
Transgenic Mice
28
Neurotransmitter Receptors
29
Proteomic Data Visualization and Future Directions
  • information overload
  • data integration
  • ease of visualization

30
Network for NMDA and glutamate receptors
31
Network for NMDA and glutamate receptors
(Zoom-in)
32
Scoring Algorithm for Spectral Analysis
33
SALSA Overview
  • SALSA is a tool for identifying MS-MS spectra in
    Xcalibur analysis files that display specific
    user-defined characteristics. Because these
    characteristics correspond to structural features
    of a peptide, SALSA allows the user to
    selectively locate MS-MS spectra of specific
    peptides or their variant or modified forms.



product ion

loss

charged
Mass difference
neutral loss


T

W

D

G

A

ion series

34
GAIIGLMGGV
GAIIGLMGG
GAIIGLMG
GAIIGLMGGVV
GAIIGLM
GAIIGL
GAIIG
GAII
Methionine Oxidation 16 amu (one oxygen atom)
GAI
GA
35
GAIIGLMVGGVV GAIIGLMVGGVV 7 amu
About PowerShow.com