An introduction to computational analysis of mass spectrometry-based proteomics data. In this video, I give a recap of the experimental data before diving into how search databases are created, how spectra are searched against these, how false discovery rates (FDRs) are estimated, and finally the challenges of inferring proteins. If you are not already familiar with MS-based proteomics, I strongly recommend that you watch my short introduction to the core concepts first: 0:00 Introduction: computational proteomics and overview of the presentation. 0:36 Experimental recap: sample preparation, tryptic digest, and MS-based proteomics. 1:05 Search database: sequences, in silico digestion, PTM expansion, and fragment ion prediction 2:20 Spectrum matching: peptide-spectrum matches (PSMs), precursor mass filter, and scoring schemes 3:23 Target-decoy search: decoy spectra, score distributions, and FDR estimation 4:44 Protein inference: equivalent proteins, subset proteins, and protein groups Thank you to David Tabb for his original slide deck that I took inspiration from ( ).