Small proteins, composed of fewer than 100 amino acids, have been historically neglected due to challenges in their annotation and biochemical detection. Despite their small size, they are crucial for essential cellular processes such as gene expression regulation, signal transduction, enzymatic activity, structural integrity, stress response, and development. In current proteomic analysis, the comprehensive identification of small proteins is hampered by signal suppression from larger proteins, their inherent low abundance, incomplete genome annotation, and inappropriate database search criteria. While ribosomal profiling and bioinformatic adjustments can overcome some impediments in genome annotation and database searches, bottlenecks in the front end of mass spectrometry (MS)-based proteomic analysis, such as sample preparation and getting rid of suppression from larger proteins in LC-MS/MS analysis, remain unsolved. To address these challenges, we developed a homemade StageTip for small protein purification before LC-MS/MS analysis, resulting in a 1.5-fold increase in small protein coverage and a 2.5-fold increase in unique peptides compared to the total proteome. In line with in silico predictions, using Lys-C instead of trypsin expanded the small protein dataset from 239 to 270 in E. coli, the largest resource so far. Further investigating the role of small proteins in the pathogenesis of Parkinson"s" diseases, a total of 26 small proteins were significantly changed between LRRK2 p.G2019S iPSCs and mutation-corrected (LRRK2 p.G2019G) iPSCs. Small proteins related to mitochondrial functions and presynaptic function were upregulated in LRRK2 p.G2019S, while stress response and protein translation pathways were enriched in corrected iPSCs. Interestingly, 6 out of these 26 small proteins were mitochondrial proteins and interacted, suggesting involvement in Complex III formation. Notably, most small proteins were annotated with membrane-bound organelle localization, consistent with their roles in regulating cellular functions through interactions with larger protein complexes. These findings underscore the feasibility of our novel approach in exploring the critical roles of small proteins in cellular physiology and disease mechanisms.