Final Ms Apps Ppt

7/30/2019 Final Ms Apps Ppt

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GC MS

LC MS/MS

SELDI-TOF

CEMS

HPTLC MS

ICPMS

TGAMS2


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Niemann-Pick C1 Disease from Human Plasma

Adrenal Cortical Dysfunction by Liquid Chromatography-Tandem Mass

Spectrometry

Cancer using SELDI-TOF MS

Propionic Acedemia using GCMS

Diagnosing Diabetes

Diagnosis of vitamin B12 deficiency

Diagnosis of neuroblastomas etc.

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Phenyalanine to Tyrosine

PKU is a metabolic disorder caused by a deficiency of the liver

enzyme phenylalanine hydroxylase

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Fig. 1. A drop of blood is spotted onto a sample card, and an aliquot is extracted

for analysis

by MS or GC-MS. The peak profile is used to diagnose metabolic errors.

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Amino acids extraction

200 L methanol/HCl 0.1% in a screw cap vial, 1 h, at 4C or bysonication for 1 min.

100 L of the extract placed in a vial,

Addition of the internal standard and removing of the reagent inanitrogen stream

AA derivatization

Esterification: 500 Lbutanol: acetyl chloride, 4:1 (v/v), 30 min, at

100C. The excess reagent is removed with a stream of nitrogen7


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Analytical instrument :

Trace DSQ ThermoFinnigan quadruple mass spectrometer

Trace GC.

Column

capillary column Rtx-5ms

30 m 0.25 mm, 0.25 m

TC Prog. : : 50 C (1 min) to 310 C, at 20 C/min.

Carrier gas : Helium (99.9995%)

Flow rate : 1 mL/min

MS

Ion source : EI mode

SIM mode

Scan m/z 50 to 5008


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Fig. 2. The mass spectra of the phenylalanine and tyrosine as trifluoroacetic

butyl ester derivatives used for PKU diagnosis

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The fingerprint chromatograms in SIM-GC-MS for a PKU patient (our results) present

the differences between the first (top chromatogram) and the third month of treatment

(second chromatogram) . The retention times for the amino acids: 15N-Gly at 6.40

min, Val at 7.02 min, Leu at 7.50 min, Pro at 8.40 min, Phe at 9.70 min and Tyr at

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PKU diagnosis could be tested by calculating the ratio Phe/Tyr using 20

L of blood spots.

A comparison of two chromatograms demonstrates the benefit of thetreatment in the case of a PKU child .

The first fingerprint chromatogram presents higher intensity of the

phenylalanine peak in the case of a patient presenting PKU, and after

three months of treatment the peak of Phe is significantly decreased.

The ratio of Phe/Tyr decreased from 2.30 at 1.02.

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What is Niemann-Pick Disease?

- Neurodegenerative disease

- metabolic dysfunction

Why LC-MS/MS?

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How is it Diagnosed?

Monitoring oxidation products of cholesterol in plasma

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Fig.2 Spectra for 7-ketocholesterol

dimethylglycinate (KC-DMG)

Fig.1 Spectra for cholestane-5-hydroxy-3,6-bis (dimethylglycinate)(CH-DMG2)


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Analysis:

- Sample preparation

Derivatization by N,N-Dimethyl glycine

-LC-MS/MS

APCI

MRM scan Mode

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What is SELDI-TOF MS ?

Difference between the mass spectrometric proteomic patterns ofserum

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Take 1-3 l serum sample

Spot sample on the activated protein chip array

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Also known as Kimmelstiel-Wilson syndrome

Renal disease

Caused by angiopathy of capillaries in the kidney glomeruli

Risk factors- elevated urinary albumin excretion, increased blood

pressure, and poor glycemic control

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To extract maximal information proteins present in smallquantities

CE-ESP-MS: Fast, sensitive, automated

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Detection of the current status of diabetic nephropathy of diabeticpatients.

Differentiation between patients with diabetes mellitus and

healthy controls in general.

Evaluation of CE-MS-defined patterns derived from urinarypolypeptides of patients with diabetic nephropathy differ fromthose of patients with other chronic renal diseases.

Making a statement of the progression of diabetic nephropathy.

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Validation of the treatment success of drugs, likecandesartan in context of diabetic nephropathy

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Analogue insulin.

Animal insulin :-bovine insulin and porcine insulin.

Human insulin

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Planar chromatography is well suited for peptide analysis.

Analysis of intact proteins.

Simple to medium complex mixtures.

Peptides from tryptic digest can be separated by HPTLC and

analyzed by MS.

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Human insulin

Bovine insulin

Porcine insulin

This were all diluted in 0.1% TFA (1mg/ml)

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HPTLC silica gel Si 60 F254 20 10 cm.

Automated TLC Sampler 4

Band length :- 5 mm

Track distance :-10 mm

Application volume :- 5 micro litre.

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Human insulin Porcine insulin Bovine insulin

968,7816[M + 6H]6+ 963,78[M+6H]6+ 956,4421 [M+6H]

1162,5367[M+5H] 1156,3347[M+5H] 1147,5288[M+H5H]

1452,6680[M+4H] 1445,1658[M+4H] 1434,1584[M+4H]

1936,5513[M+3H] 1926,5491[M+3H] 1912,2065[M+3H]

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Provide rapid, comprehensive, multi-componentanalyses

Maintain or improve sensitivity, selectivity, and

accuracy

Replacing many older traditional techniques

Is becoming an indispensable tool in clinical laboratory

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THANK YOU

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Final Ms Apps Ppt