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


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



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.


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.


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.


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.


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



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