Transcript Methodology for MALDI PTM application

Methodology for MALDI PTM application
One of the application of MALDI is for the determination of the post
translation modification of the protein, which can be done by interpretation
of cn ions signals in the mass spectrum
 Related Los: Laser properties, Matrix properties
> Prior Viewing – IDD-27. In gel digestion, IDD-29. Matrix preparation for MALDI
analysis, IDD-30. Matrix Instrumentation
Future Viewing – IDD-31. MALDI-TOF data analysis, IDD-45. Proteomic Profiling
Global
>
 Course Name: MALDI PTM application
 Level(UG/PG): PG
 Author(s) : Dinesh Raghu, Vinayak Pachapur
 Mentor: Dr. Sanjeeva Srivastava
*The contents in this ppt are licensed under Creative Commons Attribution-NonCommercial-ShareAlike 2.5 India license
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Learning objectives
After interacting with this learning object, the learner will be able to:
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Operate the MALDI instrumentation
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Analyze the mechanism behind the MALDI-MS analysis
3.
Calculate the post translation modification
4.
Assess the troubleshooting steps involved in the experiments.
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Definitions and Keywords
1. Mass Spectrometry (MS): is a analytical technique used to measure an intrinsic
property of a bio-molecule, its mass, with very high dynamic sensitivity.
2. Matrix Assisted Laser Desorption Ionization (MALDI): MALDI is effective ionization
system for generating gas-phase ions of proteins and peptides for mass spectrometric
detection. The analyte of interest is embedded in dried matrix-sample is exposed to
pulses from a UV laser for further separation and detection.
3. PTM: Post translation modification, most protein tend to get modified after the post
translation step before being functional. PTM plays an major role in making changes in
the final product of expression and contributing towards diseases and biological
processes. To name a few PTM’s
a) Phosphorylation: phosphate group addition at serine, tyrosine, threonine or histidine
amino acid residue.
b) Glycosylation: carbohydrate/glycosyl group addition at either asparagine,
hydroxylysine, serine, or threonine amino acid residue.
c) Acetylation: acetyl group addition at the N-terminus of the protein.
d) Methylation: methyl group addition at lysine or arginine residues.
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Master Layout
Sample loading
Slide 5
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Parameters setting Slide 6
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Firing of spots
Slide
7 -9
Spectrum analysis for PTM
Slide
10-15
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Step 1:
T1:Sample loading
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Description of the action/ interactivity
Take the user through the slides
of IDD-29. Matrix preparation for
MALDI analysis.
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Audio Narration
(if any)
Let user take the sample which
have been phosporylated.
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Step 2:
T2:Parameter settings
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Description of the action/ interactivity
Take the user through the slides
of IDD:30 MALDI instrumentation.
Animate the steps from the same
IDD till the firing step of sample.
Audio Narration
(if any)
Let user take the sample as
apomyoglobin as known
standardand spot it on the
MALDI plate.
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Step 3:
T3:Firing of spots
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Description of the action/ interactivity
Now let user select the actual target spot, by
right-click on yellow region, and specifing
the target spot and clicks on Fire button.
Animate initially low peaks, now let user
makes fine adjustment with 5 buttons, make
movement in camera view also, now display
more peaks with much high intensity. Let
user keep playing around to complete 100
profiles. Now highlight Store button, after
user click, let user have option to save the
data.
Audio Narration
(if any)
User must play around the spot
region to find sweet spot, where
the peaks are more in number
with high intensity. After the
100profiles user can save the
data.
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Step 3:
T3:Firing of spots
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Description of the action/ interactivity
Now user can select some other
spot and start firing like in
previous slide. In between firing,
if user likes to pause, user hav a
option like Abort, Resume. In
case the peaks obtained are not
fine, user can click on “abort” and
click on “clear data” to clear all
the data collected to re-start
again.
Audio Narration
(if any)
In between firing user have
option for abort, resume,
suspend and clear data. User
can select these options
depending on the profile data
obtained.
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Step 3:
T3:Firing of spots
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Description of the action/ interactivity
Instruct user to carry out peak
processing. Let user select
Processing> peak processing.
Display peak processing with user
controls. Let user have option for
methos selection, baseline
subraction, threshold, average
display of profiles.
Audio Narration
(if any)
In most cases the default
parameters for peak processing
are best suited. If user is not
okay with the data collected,
user can make peak processing
with the required settings.
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Step 4:
T4:Spectrum analysis
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Description of the action/ interactivity
After peak processing the data is ready to
be saved and exported. Let user select
File>Export>ASCII. A small window
display for user control must pop up. Let
the user selects the default values ad
clicks on Save as.. to open the Save As
window. Browse to your desired location,
enter a File name, and click ‘Save’. The
PMF data will be saved.
Audio Narration
(if any)
Once the PMF data is ready,
data in the excel format can be
exported, and saved.
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Step 4:
T4:Spectrum analysis
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Description of the action/ interactivity
Instruct user to calculate the difference in
mass/charge between two peaks.
Animate the peaks labeled as c1,
c2,c3...from one end (left side) and
y1,y2,y3...from other end (right side)
molecular mass. let user select any two
peaks for “c” ions or “y” ions. For example
in the figure highlight x1 and x2. let user
make a note of it.
Audio Narration
(if any)
The mass can be calculated
from any two peaks by taking
the difference and applying the
formulas.
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Step 4:
T4:Spectrum analysis
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mH: mass of hydraogen ataom
Description of the action/ interactivity
Instruct user to calculate the molecular
mass. Let user makes a difference of
theoretical mass and observed mass. Let
user tabulate the result accordingly in the
figure.
Audio Narration
(if any)
For the PTM identification, user
need to have the information of
observed mass from standard
peaks, and even the mass
value for each amino acids.
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Step 4:
T4:Spectrum analysis
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mH: mass of hydraogen ataom
Description of the action/ interactivity
Animate the highlighted row for user to
make a note. Show the difference in the
mass between the observed and
theoretical mass. Display the change of
80.11 to highlighted.
Audio Narration
(if any)
The difference in mass between
observed and theoretical,
determines the phosphate
group addition. i.e PTM has
taken place into the account on
this particular amino acid.
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Step 4:
T4:Spectrum analysis
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mH: mass of hydraogen ataom
Description of the action/ interactivity
In similar way like in previous slide, depict
to show a variation of 42Da, 43Da and
617.6Da.
Audio Narration
(if any)
The 42Da difference
corresponds to acetylation,
43Da for trimethylation and
617.6Da for Heme.
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Step 4:
T4:Spectrum analysis
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Description of the action/ interactivity
Audio Narration
(if any)
In similar way the PTM can be
identified if user has a basic
knowledge of the amino acid
mass. The difference inbetween two adjacent peak
mass helps to identify the PTM.
If the difference value adds up
to the phosphate, glycosylation,
acetylation and methylation
group determines the PTM.
Slide
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Introduction
Slide
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Tab 01
Slide 6
Tab 02
Slide
7 -9
Tab 03
Slide
10-15
Tab 04
Tab 05
Tab 06
Name of the section/stage
Interactivity
area
Animation area
In slide-11: Provide a spectrum for user to identify, y ions
and locate them accordingly?
Button 01
Button 02
Button 03
Instructions/ Working area
Credits
APPENDIX 1
Questionnaire:
Question 1
methyl group addition at _residues?
a) lysine
b) arginine
c) none
d) Both
Answer : d) Both
Question 2
acetyl group addition at the _of the protein?
a) N-terminus
b) C-terminus
c) y-terminus
d) Non of above
Answer:a) N-terminus
APPENDIX 1
Questionnaire:
Question 3
Mass of HexNAc1 + Na+ corresponds to?
a)
b)
c)
d)
282
382
161
165
Answer : a) 282
Question 4
PTM of phosphate group corresponds to mass of?
a) 75
b) 85
c) 80
d) 95
Answer: c) 80
APPENDIX 2
Links for further reading
Papers:
1.Hillenkamp F, Karas M, Beavis RC, Chait BT. Matrix-assisted laser
desorption/ionization mass spectrometry of biopolymers. Anal Chem.
1991, 63, 1193A-1203A.
2. Karas M, Glückmann
M, Schäfer J. Ionization in matrixassisted laser desorption/ionization: singly charged molecular ions are
the lucky survivors. J Mass Spectrom. 2000, 35, 1-12.
3. Hillenkamp F, Karas M. Mass spectrometry of peptides and proteins
by matrix-assisted ultraviolet laser desorption/ionization. Methods
Enzymol. 1990, 193, 280-295.
4. John Lennon and Kenneth Walsh, Locating and identifying
posttranslational modifications by in-source decay during MALDI-TOF
mass spectrometry, Protein Science ~1999!, 8:2487–2493.
Books:
 Proteomics: A cold spring harbor laboratory course manual by Andrew
J L and Joshua L, 2009.
APPENDIX 3
Summary
MALDI-MS is the best technique used for proteomics. Lot of progress in
instrumentation with additional applications continues to be made at a rapid pace.
One of the popular best application is to identify the post translation modification
by analysing the mass spectrum. One needs to have a through knowledge of all
the amino acid mass.