Practical Protein Chemistry - A. Darbre 1989

Micro-scale amino acid sequence analysis using a gas-phase peptide-protein sequencer
Discussion
Amount of sample analyzed

A representative list of the investigated samples is given in Table 17.3. Currently, the minimum amount of material required to obtain useful sequence information is 5–10 pmol (0.05–0.1 µg) for a medium-sized protein and 50 pmol (0.05 µg) for a short peptide. Sequencing heavily glycosylated Proteins, such as Erythropoietin (60% CARBOHYDRATES), has not posed any particular problems so far, although it is necessary to increase the Cleavage time for Amino Acids located both before and after the glycosylated residue in order to minimize residual amino acid levels caused by incomplete Cleavage of the preceding ones. A similar approach is taken for sequences containing Proline followed by amino acid residues with bulky side chains (Lys, Arg, Leu in Aplysia neuropeptide B).

Perhaps the most important advantage of the gas-phase Sequencer is that it makes it possible to analyze trace amounts of Polypeptides isolated by Polyacrylamide gel Electrophoresis in the presence of SDS and eluted from stained gels (melanoma Cell surface antigen, membrane phosphoprotein from Aplysia, interleukin II, or T-cell growth factor).

Class="center">Table 17.3. Peptides analyzed using the gas-phase sequencer

Ratio of identified residues to total residues

Amount

Analyzed sample

pmol

µg

Angiotensin

8/8

500

0.5

Angiotensin

6/8

50

0.05

Somatostatin

14/14

1400

2.0

Insulin B-chain

30/30

300

1.0

Aplysia neuropeptide B

31/34

500

2.0

Dynorphin

14/17

20

0.04

Myoglobin

90/153

10 000

165

Myoglobin

22/153

5

0.08

Drosophila larval cuticle protein 1a

55/166

850

15

Drosophila larval cuticle protein 3a

36/96

900

9

Membrane phosphoprotein (M 22∙103) from Aplysia b

23/200

15

0.3

Human T-cell growth factor b

15/160

25

1.0

Human histocompatibility antigen HLA-DR α-chain

49/300

700

23

Human histocompatibility antigen HLA-DR β-chain

39/240

500

13

Human erythropoietin c

28/150

100

1.6

Human melanoma cell surface antigen c

13/850

60

5.5

a Purified by isoelectric focusing in a urea-containing PAGE gel. Electrophoretically eluted from an SDS-PAGE gel stained with Coomassie Brilliant Blue. c 60% carbohydrate by weight.



Last update: 06/08/2026

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