The invention provides methods, compositions, and kits for multiplex fractionation of proteins in a sample. Protein-binding molecules, such as small epitope antibodies or small epitope aptamers, are used for multiplex fractionation of proteins in a protein containing sample. Detection of fractionated proteins may be used for characterization of proteins in a sample in applications such as expression profiling, identification and/or quantification of proteins in a sample, and identification or detection of biomarkers.