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Mass spectrometry is a very versatile technique which has become both a high-throughput and a research tool in the biosciences. In this review, methods of functional protein analysis are discussed from a perspective in clinical research. The basics of proteomics is introduced as well as the determination of post-translational modifications such as phosphorylation. The investigation of protein interactions involves the measurement of non-covalent complexes and small molecule ligands as well as bioaffinity mass spectrometry. Comparative analysis of sample profiles on specific surfaces is performed increasingly to distinguish disease-related changes.