Pooling Aqueous Humor Samples: Bias in 2D-LC-MS/MS Strategy?

The proteomic analysis of body fluids presents a major challenge in studies of human diseases. Traditional techniques for protein separation require large volumes and large amounts of protein, which may be difficult to obtain for certain fluids, such as the aqueous humor (AH). Two-dimensional liquid chromatography (2D-LC-MS/MS), adapted for peptides separation from complex protein mixtures, provides an alternative approach in proteomic analysis with a potential utility in biomarker research. We investigated several different 2D-LC-MS/MS methods for use with the AH of patients with cataract, traditionally used as a control group in studies of ocular diseases. We compared analyses of individual samples with analyses of pools of proteins or peptides, and found that the investigation strategy used strongly influenced protein identification. We identified 71 proteins related to extracellular proteins highly abundant in serum (e.g., albumin or transferrin) and involved in various functions, such as transport and metabolism, together with intracellular (myeloblastin) or organelle-specific proteins (cytochrome c). An evaluation of the advantages and disadvantages of each method suggested that individual analyses and the use of peptide mixtures should be favored as complementary techniques in the search for biomarkers in ocular diseases.