MALDI-ToF MS Method for the Characterization of Synthetic Polymers with Varying Dispersity and End Groups

This protocol provides detailed instructions and examples for sample preparation, data acquisition, and data analysis to characterize synthetic polymers with varying dispersity and end-groups using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We want to know how polymers react. For this, we use mass spectrometry, as it is a fantastic tool to study both expected reactions and other side reactions. In the last 10 years, MALDI-ToF MS has advanced to have so much more resolution and the fragmentation Within MS/MS.

We describe a wide range of reactions, including mono dispersed, moderately dispersed, and highly dispersed samples, and those with distinct isotopic resolution. For example, polymers with halogenated groups or those that are prone to metastable ion formation. To begin, prepare the analyte acquisition solution by adding the cation, analyte, and matrix stock solutions into tube A, and vortex the tube to mix.

Then add the cation, calibrant, and matrix stock solutions into tube B and mix thoroughly. Now, pipette 0.5 microliters of the cation, analyte, and matrix sample mixture from tube A onto the MALDI-ToF MS target plate, using the dried droplet method. Then pipette 0.5 microliters of the calibrant mix from tube B onto an adjacent spot on the target plate.

Record the exact row and column location of each spot for reference during data acquisition. Insert the MALDI-ToF MS target plate into the instrument and dock the target plate using the instrument control software. Select and open a method file in the instrument control software.

Choose either the reflector positive or linear positive method based on the analyte's mass range. Next, press the start button on the instrument control software to begin MALDI-ToF MS data acquisition. Allow the acquisition to complete automatically or press stop when desired.

Then click add to transfer the MALDI-ToF MS single spectrum to the sum spectrum buffer. Reposition the MALDI-ToF MS laser by viewing the camera feed and clicking anywhere within the same selected well to target a new position. Repeat the MALDI-ToF MS acquisition to continue adding spectra until the results are satisfactory.

Now, select the calibration sample on the MALDI-ToF MS target plate and perform a new acquisition following the previously described data acquisition steps. Navigate to the calibration tab in the instrument control software and open the appropriate mass control list that corresponds to the selected calibrant. Match the calibration peaks in the acquired MALDI-ToF mass spectrum with the reference masses listed in the mass control list.

In the mass control list, select the mass corresponding to the first calibration peak observed in the MALDI-ToF mass spectrum. Set the reference peak by clicking to the left of the calibrate signal so that the peak is selected. Then click apply to assign the calibrant signal as the reference mass.

Repeat the procedure for all additional calibration peaks by selecting the next mass of interest in the mass control list, aligning the corresponding calibrant peak, and applying the reference setting. For calibrated polymer analyte sample acquisition, locate and select the well containing the cation, analyte, and matrix mixture using the instrument control software. Perform MALDI-ToF MS data acquisition using the calibrated method, as demonstrated earlier.

Add an appropriate file name and save the calibrated analyte mass spectrum for further analysis. The MALDI-ToF MS spectrum of Tris G1 6 showed a single peak at 505.239 Daltons, matching its theoretical mass and confirming complete deprotection. PDMP polymers with chloride and proton end groups displayed a main peak at 859.426 Daltons, along with sodium-substituted and cyclic variants.

The full PDMP distribution spanned 600 to 1, 600 Daltons, with repeat units averaging 100.056 Daltons. Linear polyethylene brassylyate polymers exhibited alpha-hydroxy omega-propargyl, alpha-propargyl omega-propargyl, and alpha-hydroxy omega-hydroxy types in three distributions. Azide-functionalized linear polyethylene brassylyate polymers showed peaks for alpha-azido omega-azido, alpha-propargyl omega-propargyl, and alpha-propargyl omega-azido, plus the metastable ions.

Comb polymers revealed oxidation states with 16 Dalton intervals, and K plus adduct shifts confirming quadruple oxidation. Methoxy PEG, without the propargyl group 539 and 583 will remain unreactive and increase once the majority of these propargyl groups form higher molecular weights.