Evaluation of FASP for Mass Spectrometry-Based Untargeted Metabolomics Analysis of Urine Samples

Filter-Aided Sample Preparation (FASP) is a well-established method in proteomics, yet its potential for the parallel recovery of metabolites remains largely unexplored. Herein, we evaluate the performance of FASP as a straightforward workflow for the simultaneous isolation of protein and corresponding metabolite fractions from a single urine sample. The FASP-based LC-MS/MS approach for both proteomics and metabolomics analysis identified 3,163 nonredundant peptides corresponding to 957 unique protein groups. The metabolomic profile comparison of three urine fractions, specifically FASP-concentrated, FASP flow-through, and raw samples, resulted in the identification of 176 common metabolites. Next, as a proof-of-concept, the FASP protocol was applied to compare the metabolomic profiles of clinical urine samples from healthy individuals (n = 13) and patients with Ta bladder cancer (n = 12). The metabolomic modulation was consistent with previously reported findings, highlighting perturbations in phenylacetate, purine, and tryptophan metabolism, as reflected by changes in metabolites such as adenosine monophosphate (AMP), phenylacetic acid, glutamine, cytosine, and l-tryptophan. FASP protocol can be effectively adapted for the concurrent profiling of both proteomic and metabolomic fractions from urine samples. Thus, FASP-based workflow represents a viable alternative for single-step sample preparation, facilitating subsequent quantitative multiomics data integration.