Lecture 5 - Proteins MS

Question 1

What is MALDI analysis used for?

Answer 1

Intact proteins
Application to proteomics and peptidomics

Identification of:
Micro-organisms
Diseases biomarker dicovery
MALDI imaging

Question 2

Outline bacterial identification

Answer 2

1) Unknown ‘single colony’
2) Sample preparation (HCCA matrix solution)
3) Maldi-ToF MS spectra.
4) Data evaluation (identification/classification)

detection limit :
10^5 cells, aprox. 0.5ug
5x10^5 cells, 25ng

Analysis of outer surface proteins
Distinguish genetic mutants of identical strains

Question 3

Outline biomarker discovery

Answer 3

Protein profiling of the serum proteome

Universally applied e.g. Cancer, Neurological disease, Eczema, Allergy

BUT, 90% of serum is albumin

Question 4

How is albumin removed?

Answer 4

Affinity chromatography
PCT
UF (ultrafiltration)
- Kidney is good UF membrane, good for urinary biomarker discovery (

Question 5

What is Tamm-Horsfall protein (uromodulin)?

Answer 5

Abundant soluble urinary protein

Question 6

What is renal tubulopathies?

apical membrane proteins

Answer 6

disorders that affect function of renal tubule epithelia.

Exosomes only account for around 3% of the total protein urine isolation

Question 7

Outline biomarker : magnetic beads

Answer 7

Used for immunoprecipitation

Metallic proteins are precipitated using magnets

Question 8

What is the difference between SELDI and MALDI?

Answer 8

SELDI: Some proteins in the sample bind to the surface, while the others are removed by washing. matrix is applied to the surface and allowed to crystallize with the sample peptides

MALDI: protein or peptide sample is mixed with the matrix molecule in solution and small amounts of the mixture are deposited on a surface and allowed to dry

Question 9

What MS techniques are used for protein identification?

Answer 9

MALDI-ToF MS
- Peptide mass fingerprinting

Electrospray MS
- HPLC-MS/MS

Question 10

Outline proteomics analysis protocol

Answer 10

1) Isolate protein of interest
2) Digest protein into corresponding peptides (proteolytic method)
3) Analyse peptides formed by MS to identify the protein

Question 11

What is Sodium Dodecyl sulfate used for?

Answer 11

Polyacrylamide Gel electrophoresis

It transfers a charge into proteins

Question 12

What is 2D Page?

Answer 12

Isoelectric focuing (IEF)

Utilizes electrophoretic mobility which is dependant on pH.

Proteins migrate towards their isoelectric point

Question 13

What is the digestion site of Trypsin?

Answer 13

Arginine (R) and Lysine (K)

Question 14

What is the digestion site of Chymotrypsin?

Answer 14

Tyrosine (Y) and Phenylalanine (F)

Question 15

What is the digestion site of Endo Asp N?

Answer 15

(Aspartate) D and Cysteine (C)

Question 16

What is the digestion site of Endo Glu C?

Answer 16

Glutamate (E) and Aspartate (D)

Question 17

What is the commonly used digestion agent?

Answer 17

Trypsin

Digestion is enhanced by DTT and iosoacetamide

Protocol: 16hrs, 24 or 37’c

Question 18

Outline Peptide mass fingerprinting MALDI ToF MS

Answer 18

1) Digestion of proteins
2) Calibration by trypsin autolysis peaks
3) ~10ppm required
4) Database used to match peptides found

Question 19

What is Calibration theory?

Answer 19

Measures the Accuracy and precision

Precision: Measure of repeatability

Accuracy: Proximity of obtained values to the “true” value.

These values are measured in ppm

100 ppm = 0.01%
10 ppm = 0.001%
1 ppm = 0.0001%