PAMAM-Functionalized Paper Boosts Sensitivity of Protein Analysis

A research team at Vancouver Island University in Canada has developed a new paper substrate that significantly increases the sensitivity of protein analysis by paper spray mass spectrometry (PS–MS). The paper surface has been functionalized with polyamidoamine (PAMAM) dendrimers, which can electrostatically interact with negatively charged residues on protein surfaces, as well as promote hydrogen bonding with protein surface oxygens, making PAMAM a useful material for protein extraction.

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PS–MS is a fast, inexpensive, and easy-to-use technique for analyzing small molecules and proteins in biofluids. PS–MS uses a paper substrate to collect analytes from biofluids and an electrospray source to ionize and transfer them into the mass spectrometer. The technique can detect proteins and peptides with high sensitivity and specificity, making it useful for clinical diagnostics and personalized medicine. PS–MS has the potential to revolutionize disease diagnosis and treatment by enabling the rapid and accurate detection of biomarkers.

The team used PAMAM-functionalized PS–MS paper strips to extract proteins from biofluids, dried them, and then measured them with PS–MS. The use of PAMAM-functionalized paper substrates provided significantly greater sensitivity than unmodified paper strips: sixfold greater sensitivity for albumin, 11-fold for hemoglobin, sevenfold for insulin, and twofold for lysozyme.

PAMAM-functionalized PS-MS paper strips are a novel substrate for protein analysis by PS–MS. They are paper strips that have been functionalized with PAMAM dendrimers, which are a branched polymeric compound with an ethylenediamine core linked to repeating PAMAM units. The PAMAM dendrimers generate an outer surface rich in primary amines that can electrostatically interact with negatively charged residues on protein surfaces and promote hydrogen bonding with protein surface oxygens, making PAMAM a useful material for protein extraction. By using PAMAM-functionalized paper substrates for PS–MS, protein analysis becomes more sensitive, enabling the detection of lower concentrations of disease-related proteins in biofluids.

The team evaluated the analytical performance of the functionalized paper substrate by analyzing albumin in urine. The method achieved linearity with R² > 0.99, LOD of 1.1 μg/mL, LOQ of 3.8 μg/mL, precision better than 10%, and relative recovery 70–83%. The method was then applied to quantify urinary albumin from nine anonymous patient samples, with concentrations ranging from 6.5 to 77.4 μg/mL, illustrating its potential for the diagnosis of microalbuminuria.

The research demonstrates the utility of paper modification with the PAMAM dendrimer for sensitive PS–MS analysis of proteins, opening a path for further applications in clinical diagnosis through the analysis of disease-related proteins. The team's findings have been published in the journal Analytical Chemistry (1).

The new paper substrate could revolutionize the way researcher’s analyze proteins, particularly in clinical diagnosis. The sensitivity achieved by PAMAM-functionalized paper could enable detection of much lower concentrations of disease-related proteins in biofluids, potentially leading to earlier and more accurate diagnoses of diseases.

Reference

(1) Pereira, I.; Monaghan, J.; Abruzzi, L. R.; Gill, C. G. PAMAM-Functionalized Paper as a New Substrate for the Paper Spray Mass Spectrometry Measurement of Proteins. Anal. Chem. 2023. DOI: https://doi.org/10.1021/acs.analchem.2c05316