Investigation of the human tear film proteome using multiple proteomic approaches

Mass Spectrometry and Proteomics Facility, The Ohio State University, Columbus, OH 43210, USA.
Molecular vision (Impact Factor: 1.99). 02/2008; 14:456-70.
Source: PubMed


The purpose of this work was to examine the tear film proteome using a combination of one-dimensional (1D) and two dimensional (2D) gel electrophoresis and mass spectrometry-based techniques and to explore the effect of the tear collection methods on the tear proteome.
Tear samples from eight normal non-contact lens wearing human subjects collected by Drummond glass microcapillary and Schirmer strips were subjected to 1D-sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 2D-SDS-PAGE, and 2D LC-MS/MS (Multidimensional protein identification technology - MudPIT). Bands or cores from the 1D- and 2D-SDS-PAGE were cut, digested with trypsin, and analyzed by tandem mass spectrometry for identification by the generation of sequence tags.
In total (across sampling and proteomic methods), 97 unique proteins were observed, and a significant number of the spots/bands in the PAGE were from posttranslational modifications. Fifty-four unique proteins were identified from proteins extracted from the Schirmer strips in comparison to 13 unique proteins identified from capillary tubes, and 30 unique proteins were identified by both collection methods. Secreted (serum) proteins were predominantly observed from tears collected by capillary whereas a combination of cellular and serum proteins were identified from tear film collected by Schirmer strips.
Overall, these results suggest that the tear film collection and the proteomic method impacts the proteins present in the tear film and that care should be exercised in choosing a tear collection method to best correlate to the experiment being conducted or the hypothesis that is being tested.

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    • "Compared to serum, the protein concentration in tear fluid in general is very low [87], which can also lead to problems in terms of analysis. Nevertheless, a large number of proteins are unique to tear fluid [64]. Therefore, sensitive and accurate MS techniques are the method of choice for its analysis [88] (Table 1). "
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    • "Ammonium bicarbonate has been previously used for protein extraction from Schirmer strips [15] [17]. For instance, Green-Church and coworkers used ammonium bicarbonate to extract proteins from Schirmer strips and then precipitated the extract with acetone [17]. Because tear fluid contains NaCl, adding NaCl to the extraction buffer may increase the solubility of tear proteins and promote their extraction from the strips [18]. "
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    • "Recently, Zhou et al. (2012) reported the presence of 1543 various proteins in tear samples through a non gel-based technique. However, to our knowledge patterns obtained in 2D gel electrophoresis could not suggest more than 250 proteins in normal tear proteomes (de Souza et al., 2006; Green-Church et al., 2008; Herber et al., 2001; Saijyothi et al., 2010). This significant difference originates from the nature of gel-based techniques which always have lower detectability in contrast to gel-free (LC-MS) methods. "
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