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Investigation of the human tear film proteome using multiple proteomic approaches.

Green-Church KB, Nichols KK, Kleinholz NM, Zhang L, Nichols JJ - Mol. Vis. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Mass Spectrometry and Proteomics Facility, The Ohio State University, Columbus, OH 43210, USA. green-church.1@osu.edu

ABSTRACT

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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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Related in: MedlinePlus

Eighteen-centimeter 2D-SDS–PAGE stained with SyproRuby of capillary-collected tears. The gel is labeled with subsequent protein identifications by nano-LC/MS/MS.
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f8: Eighteen-centimeter 2D-SDS–PAGE stained with SyproRuby of capillary-collected tears. The gel is labeled with subsequent protein identifications by nano-LC/MS/MS.

Mentions: Figure 7 and Figure 8 are the individual SyproRuby stained gels labeled with the protein identifications. Similar to other published work [10,45,51,52], as many as 500 protein spots are observed in the 2D gel. 2D electrophoresis is not the most efficient way to identify all the proteins in a complex mixture of proteins. Rather, it is better suited to examine protein pattern changes between two samples. In this case, 2D gel electrophoresis was mainly used to examine pattern changes observed between capillary- and Schirmer strip-collected tear films. Protein identifications were conducted on 58 selected spots that were cored, digested, and analyzed by nano LC-MS/MS. A total of 31 unique proteins were identified, and 27 of the 58 spots matched proteins identified from other cores in other regions of the gel, similar to previous results (e.g., the multiple spots along the 80 kDa region are predicted to be glycosylated lactoferrin).


Investigation of the human tear film proteome using multiple proteomic approaches.

Green-Church KB, Nichols KK, Kleinholz NM, Zhang L, Nichols JJ - Mol. Vis. (2008)

Eighteen-centimeter 2D-SDS–PAGE stained with SyproRuby of capillary-collected tears. The gel is labeled with subsequent protein identifications by nano-LC/MS/MS.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2268847&req=5

f8: Eighteen-centimeter 2D-SDS–PAGE stained with SyproRuby of capillary-collected tears. The gel is labeled with subsequent protein identifications by nano-LC/MS/MS.
Mentions: Figure 7 and Figure 8 are the individual SyproRuby stained gels labeled with the protein identifications. Similar to other published work [10,45,51,52], as many as 500 protein spots are observed in the 2D gel. 2D electrophoresis is not the most efficient way to identify all the proteins in a complex mixture of proteins. Rather, it is better suited to examine protein pattern changes between two samples. In this case, 2D gel electrophoresis was mainly used to examine pattern changes observed between capillary- and Schirmer strip-collected tear films. Protein identifications were conducted on 58 selected spots that were cored, digested, and analyzed by nano LC-MS/MS. A total of 31 unique proteins were identified, and 27 of the 58 spots matched proteins identified from other cores in other regions of the gel, similar to previous results (e.g., the multiple spots along the 80 kDa region are predicted to be glycosylated lactoferrin).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Mass Spectrometry and Proteomics Facility, The Ohio State University, Columbus, OH 43210, USA. green-church.1@osu.edu

ABSTRACT

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus