Single-cell proteomics using mass spectrometry.

Over the past 2 to 3 years, mass-spectrometry-based single-cell proteomics (SCP) has experienced transformative improvements in microfluidic and robotic sample preparation, innovative MS1- and MS2-based multiplexing strategies, and specialized hardware (e.g., timsTOF Ultra 2, Astral), which have dramatically boosted sensitivity, throughput, and proteome coverage from picogram-level protein inputs. Concurrently, tailored computational workflows that encompass normalization, imputation, and no-code platforms have addressed pervasive missing data challenges and standardized analyses, collectively enabling high-throughput, reproducible profiling of cellular heterogeneity. This minireview summarizes the latest progress in SCP technology and software solutions, highlighting how the closer integration of analytical, computational, and experimental strategies will facilitate a deeper and broader coverage of single-cell proteomes.