Multifunctional ZrO(2) nanoparticles (NPs) and ZrO(2)-SiO(2) nanorods (NRs) have been successfully applied as the matrices for cyclodextrins and as affinity probes for enrichment of peptides (leucine-enkephalin, methionine-enkephalin and thiopeptide), phosphopeptides (from tryptic digestion products of beta-casein) and phosphoproteins from complex samples (urine and milk) in atmospheric pressure matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and MALDI time-of-flight (TOF) MS. The results show that the ZrO(2) NPs and ZrO(2)-SiO(2) NRs can interact with target molecules (cyclodextrins, peptides, and proteins), and the signal intensities of the analytes were significantly improved in MALDI-MS. The maximum signal intensities of the peptides were obtained at pH 4.5 using ZrO(2) NPs and ZrO(2)-SiO(2) NRs as affinity probes. The limits of detection of the peptides were found to be 75-105 fmol for atmospheric pressure MALDI-MS and those of the cyclodextrins and beta-casein were found to be 7.5-20 and 115-125 fmol, respectively, for MALDI-TOF-MS. In addition, these nanomaterials can be applied as the matrices for the analysis of cyclodextrins in urine samples by MALDI-TOF-MS. ZrO(2) NPs and ZrO(2)-SiO(2) NRs efficiently served as electrostatic probes for peptide mixtures and milk proteins because 2-11 times signal enhancement can be achieved compared with use of conventional organic matrices. Moreover, we have successfully demonstrated that the ZrO(2) NPs can be effectively applied for enrichment of phosphopeptides from tryptic digestion of beta-casein. Comparing ZrO(2) NPs with ZrO(2)-SiO(2) NRs, we found that ZrO(2) NPs exhibited better affinity towards phosphopeptides than ZrO(2)-SiO(2) NRs. Furthermore, the ZrO(2) and ZrO(2)-SiO(2) nanomaterials could be used to concentrate trace amounts of peptides/proteins from aqueous solutions without tedious washing procedures. This approach is a simple, straightforward, separation-and washing-free approach for MALDI-MS analysis of cyclodextrins, peptides, proteins, and tryptic digestion products of phosphoproteins.