In the last decade, there has been a dramatic progress in separation techniques, mass spectrometry, and bioinformatics, and this progress has significantly improved the techniques on protein analysis. However, the analysis of low-abundance proteins is still challenging because of the limited performance in the method of choice compared to the complexity and the vast dynamic range of biological samples. Since this issue is a big obstacle in most proteomics investigations, great interest has been paid recently to various techniques, such as multi-dimensional analysis, specific peptide selection, high-abundance protein depletion, ligand library treatment, to address this challenge. Therefore, here, the author reviews recent nano liquid chromatography coupled with tandem mass spectrometry-based studies on the deep proteome, mainly focusing on their methods and perspectives.