The sequencing of the genomes of many different species has greatly helped our understanding of organelles. This information has driven the development of mass spectrometry (MS)-based methods for large-scale analysis of proteins. One of these methods uses two-dimensional liquid chromatography (2DLC) coupled on-line to tandem MS and is described here. In this method, proteins are first proteolytically digested, and then the peptides are separated in two dimensions. Typically, separation in the first dimension is based on charge interactions with a strong cation exchange (SCX) resin, whereas separation in the second dimension is based on hydrophobic interactions with a reversed-phase (RP) support. Peptides are eluted from the SCX resin using increasing concentrations of a salt and subsequently trapped on the RP resin. Next, the salt is washed from the system, and the peptides are eluted using an increasing gradient of a non-polar organic solvent. Eluting peptides are mass analyzed and fragmented to generate tandem mass spectra. These tandem mass spectra can be used to search sequence databases to identify peptides by matching amino-acid sequences to each spectrum.