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A pilot protein profiling of one of the smallest organism ureaplasma urealyticum, a pathogen causing urogenital and placental infection/inflammation, using the new protein fractionation system of Beckmen Coulter’s ProteomeLab PF 2D is reported here. ProteomeLab PF 2D is a two-dimensional liquid chromatographic system to fractionate complex proteomes. This system consists of a chromatofocusing column (250 × 2.1 mm) to separate the protein based on charge/pI and a nonporous reversed-phase column (4.6 × 33 mm) to separate the protein based on hydrophobicity. The advantages of this technique include high loading capacity and better detection of hydrophobic and extreme PI and molecular weight proteins. Up to 1000 protein fractions are collected. So far, 30 proteins such as GroEL, heat shock protein, protein L7/L12, and others have been identified using MALDI-TOF or FT-ICR MS with Sequest or Mascot search engines. This study has demonstrate that ProteomeLab PF 2D with mass spectrometry and some precautions is a potentially powerful tool to identify proteins from a large proteome.