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This lecture covers the fundamentals of protein mass spectrometry and proteomics, starting with an introduction to chromatography techniques and the history of chromatography. It delves into the basis of chromatography, efficiency, resolution, and factors affecting chromatographic performance. The lecture also discusses the importance of selectivity, efficiency, and resolution in chromatography, as well as the concept of theoretical plates. Additionally, it explores the application of peptide isoelectric focusing (IEF) in mass spectrometry data validation and filtering, including the use of immobilized pH gradient (IPG) strips. The lecture concludes with a discussion on the separation efficiency, visualization, and practical applications of off-gel electrophoresis in proteomics.