MALDI-TOF

How is a MALDI-TOF measurement made?

The sample is mixed with a large excess of matrix, a small organic compound chosen because it absorbs strongly at the laser's wavelength, and dried onto a metal plate. A pulsed laser hits the spot; the matrix absorbs the energy and carries the peptide molecules into the gas phase as ions.

A review of quantitative MALDI describes a typical preparation as a sample around 10 µM pre-mixed with matrix around 10 mM, with about 1 µL applied to the plate and dried to a heterogeneous crystalline surface (Duncan and colleagues, Briefings in Functional Genomics and Proteomics, 2008). The ions are accelerated into a flight tube, and since lighter ions arrive first, flight time converts to mass.

Why do MALDI spectra look simpler than LC-MS spectra?

Electrospray, used in LC-MS, tends to put several protons on a peptide, so one molecule appears as a ladder of charge states. Duncan and colleagues contrast this with MALDI's tendency to yield singly charged ions, which helps with mixtures because each molecule shows up roughly once, at its mass plus one proton.

That simplicity suits a quick identity screen: read the [M+H]+ peak and compare it with the expected value. A 2016 set of recommendations for peptides used in mass spectrometry assays lists a MALDI or ESI spectrum for mass identity confirmation, alongside an HPLC chromatogram for purity and amino acid analysis for concentration (Hoofnagle and colleagues, Clinical Chemistry, 2016).

How does MALDI-TOF compare with LC-MS for peptide identity?

Why is MALDI-TOF a weak basis for purity or amount?

The main issue is reproducibility. Matrix and sample do not crystallise evenly, and Duncan and colleagues describe signal variation between consecutive laser shots at the same position, between positions on the same spot, and between identical loadings on different targets. The 2016 recommendations put it directly: relative ion intensities in MALDI will not accurately measure the molar content of contaminants. Quantitative MALDI is possible with internal standards and careful method work that a routine identity screen does not include.

Duncan and colleagues also note that the vast molar excess of matrix swamps the low m/z region, which is generally not shown below about m/z 500, so low-mass species such as counter-ions cannot be read from the spectrum.

A 2022 review in ACS Measurement Science Au describes time-of-flight as a common high-throughput, middle to high resolution analyser to couple with MALDI (ACS Measurement Science Au, 2022). That is enough to tell the labelled compound from a clearly different one; a one-residue change or small modification is better handled by high-resolution MS after a separation step.

Where does MALDI-TOF fit in a regulatory identity package?

The European Medicines Agency's draft guideline on synthetic peptides notes that variants of MS can be used to determine the molecular mass of a peptide and to confirm its sequence, and recommends at least two orthogonal methods for identification at release (EMA draft guideline on synthetic peptides, 2023). A MALDI mass can be one of those strands. On its own, it shows the right-sized molecule is present somewhere in the spot, not that it is the main component of the vial.

Does Tested.ph offer MALDI-TOF?

No. Identity in the Tested.ph standard package is by LC-MS, which ties each mass to a chromatographic peak. This page is for reading certificates from elsewhere that quote MALDI-TOF; our guide to reading a peptide COA shows where the identity line usually sits.