Problem BAT

8/8/2019 Problem BAT

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Problem:

Submitted to:

Mr. Ravikant Pathak

Department of Biotechnology

LHST, LPU

Submitted by:

Sunil Nagpal

Roll no. A7703-01

Reg no. 7040070056

B.tech(H)M.tech

Biotechnology



Suppose you are given Haemoglobin molecules for determination of molecular mass.

Device a scheme or experiment using mass spectroscopy, which can deliver best results.

You can take any standard MS data from the journals or laboratory sites in the web.

Whenever you are using a technique, do write why you have preffered that instead of the

other techniques.

Introduction:

Before we device any technique or experiment, it is essential to keep in mind the following

characteristics of Haemoglobin molecule, which should match with the results of our

experiment:

1. Haemoglobin is an assembly of four globular protein subunits: 2 alpha abd 2 beta chains.

2. The subunits are structurraly similar and of about same size.

3. 2 alpha chains are 146aa long each and 2 beta chains are 141aa long each.

Experiment proposed:

The author suggests parallel running two mass spectroscopy experiments and then merging their

interpretations together, to have an accurate estimation of the molecular mass of Haemoglobin.

Layout depiction:

Experiment 1 Experiment 2

Electron Spray Ionization

Ion trap mass analyser

m/z values

known m/z specific ions

detected by detector

Fast Atomic Bombardment

Ion trap mass analyser Ion trap mass analyser

m/z values

known m/z specific ion

detected by detecNo fragments.

Three-four

peaks possible

Partial Fragments

Multiple peaks

corresponding to

fragments



Steps worth consideration in both experiments:

1. Sample preparation(ESI):

Purified Hb is extensively dialyzed against distilled water and lyophilized. 1 mg of each of these

it was dissolved in distilled water (concentration 10 μg/μl). Then 10-μl aliquots of this solutionwas diluted with 20 μl of 1% formic acid, 20 μl of distilled water, and 50 μl of acetonitrile to

make the working solution for the analysis.

2. Sample preparation(FAB):

Hb is mixed with a viscous matrix of glycerol or any relatively involatile viscous matrix

and this Hb-matrix mixture is ready to be placed on FAB probe for bombardment.

3. Sample used should be less, because of the fact that both techniques are highlysensitive and thus excess of substrate may lead to haphazard of peaks and

instrument clogging.

4. Adviced sample input:1-10mol mm-3.

5. Why ion trap analyser?

• Most rapid analyser.

• More than one ion analysed at single time, unlike the other analysers like Quadrupole

which analyse only one ion at a time.

• Highly sensitive and accurate: 0.01% mass accuracy.

• Accurate molecular mass determination upto 100,000 Da.

6. Why not TOF?

• The problem of Post Source Decay associated with TOF is invariably undesired.



Justifying the two experiments:

Aim of Experiment 1:

Experiment 1 involves the use of Electron Spray Ionisation(ESI) technique, so it aims at

obtaining un-dissociated whole molecule’s m/z value to be analysed.

Considerations of experiment 1:

• Not every peak corresponds to singly charged ions of the molecule.

• Some peaks may correspond to ions of multiple charge, and hence small m/z values.

• None of the peaks is of any fragmentation.

• If the four peptide chains in Hb have separated, then peaks corresponding to each chain

may also be there.

Why used ESI?

Highly efficient technique to ionize the sample without inducing any fragmentation. Electron

impact and Chemical ionization lead to fragmentation, and this is undesired in our experiment 1.

Why not MALDI?

This technique stands expensive, as far as the instrumentation of Laser, camera, Teflon plates is

considered. But importantly, it needs to be associated with TOF for better results, where the

problem of Post Source Decay(PSD) arises, and this is not desired at all.

Aim of experiment 2:



It involves the use of FAB, so it aims at getting partially fragmented sample, each fragment

acting as a pseudomolecular ion, as either (M+H)+ or (M-H)-.

Cons iderations of experiment 2:

•

The fragmentation is not extensive.

• It is much probable that the four chains may dissociate here.

• It is much probable that each chain may get partially fragmented.

Why FAB?

Partial fragmentation is the key requirement of the second experiment, and the most reliabletechnique for it is—FAB. Where as Electron impact goes to the atomic level of fragmentation,

which will be too much extensive and Chemical ionization is also high enough not to be

considered.

Explaining the analytical procedure(Merging the two experiments):

Case 1: The four chains didn’t separate in the sample:

Data: [source: www.biochem.org]

ESI: 15300 and 31200

FAB-Ion trap:13220, 14505, 15805, 15981

Interpretation:

Documents

Problem BAT