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Home > Peptide Sequencing Techniques > Peptide Analysis
Peptide Analysis
The results of analyses of aminoacids obtained from acid or alkaline hydrolyses (see topics COAC and COAL) and from exopeptidase digestions (see topics AMPM, CPPA and CPAB) are reported in µ-moles; chemical fragmentations occasionally generate single aminoacids which are also reported thus.
All such analytical results are reported with a precision of ~ 0.01 µ-mole; however the accuracy of the result is only ~ 0.1 µ-mole; aminoacids in quantities less than 0.1 µ-mole are not reported.
The error range of 0.2 µ-mole means that at least 0.4 µ-mole of should be used in acidic or alkaline hydrolyses of shorter fragments (up to 4 aminoacids) and at least 0.7 µ-mole for peptides longer than 10 to 12 aminoacids, otherwise the results become difficult to interpret.
In exopeptidase digestions, where interpretation depends on the difference in quantity of the aminoacids, a minimum of 1.0 µ-mole is needed.
In fragmentations, results are reported in mg to the nearest 0.1 mg.
DBSL: Determination of N-terminus using dabsyl chloride.
This extremely sensitive test requires the minimum quantity of sample, 0.1 mg (default quantity). Acidic conditions will cause the decomposition of acid-sensitive tryptophan; neither can they remove an acetylating group from the N-terminus. In these 2 cases (Trp or Ace), the test gives a null result.
The result of the determination of the N-terminus with dabsyl chloride is a qualitative result only.
N-Terminal Amino Acid Analysis
Except when it is already blocked by formylation, acetylation, pyroglutamic acid formation or other chemistry, the N-terminal amino acid of proteins can be labeled with a variety of fluorescent and chromophoric reagents from Chapter 1. However, only those functional groups that survive complete protein hydrolysis, such as sulfonamides, are useful for N-terminal amino acid analysis. Dansyl chloride (D21) and dabsyl chloride (D1537) are the most commonly employed reagents for such analyses.
http://www.probes.com/handbook/sections/0905.html
COAC: Hydrolysis of a peptide using 6M HCl at 105 °C.
The quantity required is 0.4 µ-mole for short chains (4 aa's), up to 0.8 µ-mole for longer chains (> 10-12 aa's).
The quantity in mg can be estimated:
- for the original peptide from the knowledge that the issued quantity is about 10 µ-mole
- for fragments from a calculation involving the FW of the parent peptide and the standard 80% (molar) recovery rate in fragmentations.
The completeness of a hydrolysis increases with time, with some aminoacids (eg Leu, Val, Ile) being hydrolysed particularly slowly.
Unfortunately, many aminoacids decompose in an acidic medium; Trp very rapidly, Cys, Met, Thr and Tyr somewhat slower. With prolonged hydrolysis times, the quantity of such aminoacids diminishes.
The rapid decomposition of Trp means it cannot be detected by acidic hydrolysis.
The default time of 24 hr is a minimum.
COAL: Hydrolysis of a peptide using barium hydroxide at 100 °C.
For quantity to use, see COAC.
As in COAC, a balance between hydrolysis and decomposition rates determines the reliability of analysis figures. In alkaline conditions, Trp is more stable; however Arg and Cys are decomposed so rapidly in alkaline conditions that they are detected very rarely.
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