Navigation Links
Direct Monitoring of Solid-Phase Peptide Synthesis by MALDI-TOF MS

Overview

Mass spectrometry has proven to be a valuable tool in verifying and improving peptide synthesis. Matrix Assisted Laser Desorption Ionization (MALDI) Time-of-Flight (TOF) MS is particularly useful in that it is rapid, easy to use and requires relatively little maintenance. This work shows the ability of MALDI-TOF MS to follow the progress of solid phase peptide synthesis. This work was first presented by A. Falick, G. Tarr and N. Sole at ABRF 96.

Key Features

Identification of peptide synthesis products

Ability to track reactions over time

Mass accuracy in DE linear mode

Method

PEPTIDE SYNTHESIS

Linear syntheses were carried out using Fmoc chemistry in the C to N direction on a PerSeptive Biosystems 9050 Plus continuous flow synthesizer. Coupling times were 1 hour, using 4 equivalents of amino acid/ activator and 8 of base. HATU, DIEA and DMF were used as solvents.

MASS SPECTROMETRY

Samples were analyzed on a Voyager-DE STR Workstation in linear mode. The laser was set at 337 nm and 50-100 laser shots were accumulated per spectrum.

Peptides were deprotected with 95% TFA at room temperature for the times noted. 1 mg of resin was treated with 20 μl of TFA, then a 1μl aliquot was removed and added to 50 μl of water. 1 μl aliquot of this diluted sample was mixed with 4 μl of a 10 mg/ml solution of alpha cyano 4-hydroxy cinnamic acid in 60% ethanol and 40% acetonitrile. A 0.5 μl droplet was placed on the MALDI target, air-dried and the target inserted into the instrument.

Results

Figure 1 shows the progress of the synthesis of the 21 mer peptide fragment from the targetting sequence of the precursor of mi tochondrial aspartate aminotransferase, ALLQSRLLLSAPRRAAATARA. A deprotection time of 1hour was used for these experiments. Had there been an issue with the synthesis, the exact problem step would have been diagnosed.

Figure 2 shows some of the minor peaks in the spectrum taken after step 13. Small amounts of deletion peptides are seen in addition to small peaks representing residual Pbf (Arg-protecting group). These are due to released Pbf attacking, for example, the Serine residue. Small contributions from cationization with sodium and potassium are also observed.

Figure 3 shows the MH+ region of the completed and deprotected peptide. Neurotensin (MW 1,673) and ACTH 18-39 (MW 2,465) were used as internal calibrants. The average error of the mass measurement was +/- 0.03 Da (5 replicates). This measurement is more than would be required to differentiate between a carboxyl and an amide (one Da difference). All masses are monoisotopic since the resolution of the instrument was sufficient to resolve isotopes at these masses.

1 hour deprotection is not required to determine if the synthesis is proceeding correctly. Figure 4 shows the peptide data after step 10 with a 10 minute deprotection step. The Pbf protecting group on Arg is clearly still present but some fully deprotected product (Fmoc peptide) is visible. The t-Bu protecting group on Thr is completely absent. No t-Bu group was detected after > 5 minute treatment with TFA. The trityl group used to protect Gln behaves similarly.

The result in Figure 5 is from the deprotection of the synthesis of the peptide SEHFLANES-OH. In this case the deprotection step was for 5 minutes only, and the presence of trityl and up to 3 t-butyl groups can be detected.

The full time course experiment up to 1 hour is seen in Figure 6. After 1 hour the unprotected peptide is the principal peak in the spectrum.

Conclusion

MALDI-TOF MS is a rapid and easy way of monitoring peptide synthesis. Small amounts of resin can quickly be treated with TFA and the peptides analyzed with no further treatment or purification. Mass measurement can be carried out on crude samples and be used to verify identity before final purification. Removal of protecting groups can be followed as a function of time and incomplete coupling can be identified very quickly.

Acknowledgements

PerSeptive Biosystems would like to acknowledge Arnold Falick and Keith Waddell of PE Biosystems for their contributions to this application note.


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Counting Colonies or Plaques Directly from Images of Agar Plates
2. High-Efficiency Site-Directed Mutagenesis of Large Plasmid Vectors
3. Constructing Directional cDNA Libraries from Limited Amounts of RNA
4. Generate High-Quality, Directional Plasmid cDNA Libraries
5. Optimizing the Direct Amplification of Missing cDNA 5 Ends Using the Eppendorf Mastercycler gradient
6. Optimizing the Direct Amplification of Missing cDNA 5 Ends Using the Eppendorf Mastercycler gradient
7. TripleMaster PCR System: Direct Comparison with Competitors
8. DirectPrep 96 Miniprep System for cost-effective, high-throughput plasmid DNA purification
9. A Further Step in Understanding Apoptosis Direct Detection of PARP Cleavage
10. YOUR DATA: Direct Real-time RT-PCR on Sympathetic Neuron Lysates Silencing of Focal Adhesion Kinases with Silencer Validated siRNAs
11. YOUR DATA: Direct Real-time RT-PCR on Sympathetic Neuron Lysates Silencing of Focal Adhesion Kinases with Silencer Validated siRNAs
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:2/11/2016)... Inc. (NASDAQ: NBIX ) today announced its financial results for ... --> --> For the fourth quarter of ... $0.34 loss per share, compared to a net loss of $19.4 ... 2014. For the year ended December 31, 2015, the Company reported ... as compared to a net loss of $60.5 million, or $0.81 ...
(Date:2/11/2016)... , Feb. 11, 2016  Wellcentive today announced ... Portland, Oregon -based community care organization ... population health analytics, quality reporting and care management ... strengthen its team of quality managers, analysts and ... the provider groups serving FamilyCare members. ...
(Date:2/11/2016)... ... February 11, 2016 , ... ... 150 years, continues today to pursue the highest level of accuracy and quality ... the AR9 Refractometer and the AR5 Refractometer. Accurate, reliable and tough enough ...
(Date:2/10/2016)... Md. , Feb. 10, 2016  The Maryland ... Busch , has announced that University of Maryland School ... PhD, MBA and University of Maryland Medical System President ... of the "Speaker,s Medallion," the highest honor given to ... of Delegates. Dean Reece and Mr. ...
Breaking Biology Technology:
(Date:2/2/2016)... Technology Enhancements Accelerate Growth of X-ray Imaging ... and computed radiography markets in Thailand ... Indonesia (TIM). It provides an in-depth analysis ... as regional market drivers and restraints. The study offers ... market attractiveness, both for digital and computed radiography. Market ...
(Date:2/1/2016)... Feb. 1, 2016  Today, the first day of ... plans to develop a first of its kind workplace ... IBM Watson. In the first application of ... IBM ), and Welltok will create a new ... with cognitive analytics, delivered on Welltok,s health optimization platform. ...
(Date:1/25/2016)... Unisys Corporation (NYSE: UIS ) today announced the implementation ... New York City , to help U.S. ... the United States using passports that are ... of the system at Dulles last year. The system will ... 2016. --> pilot testing of the system at ...
Breaking Biology News(10 mins):