To optimize MALDI protocols, we changed the concentration of analytes, the solvent, the matrix:analyte ratio and deposition structure (as exemplified in Fig. for BSA-AuNCs). First, MALDI-MS with different concentrations of nanoclusters were investigated, ranging from 6.7 μM, 10 μM to 33.3 μM (Figure S3A). Then, different solvents used for both nanoclusters and matrix were investigated, with water-acetonitrile volume friction of 70/30 or 50/50, adding TFA or not (Figure ). Different methods were compared for the deposition, ranging from the co-deposition of a mixture of the NCs and matrix solutions, to the depositions of different layers alternating the matrix and the NCs solutions (Figure ). Besides, we recorded the MALDI-MS spectra with different volume ratio of matrix to analyte (from 1:1 to 10:1) as shown in Figure .