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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

1D-SDS–PAGE of coomassie stained lactoferrin and serum albumin. Shown is a graph of gel slice versus Mowse score of lactoferrin and serum albumin to determine what protein is the dominating factor from the gel band.
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f5: 1D-SDS–PAGE of coomassie stained lactoferrin and serum albumin. Shown is a graph of gel slice versus Mowse score of lactoferrin and serum albumin to determine what protein is the dominating factor from the gel band.

Mentions: The sequence coverage observed in the mass spectrometry experiments reflects the amount of the protein. Highly abundant proteins yield high sequence coverage while low abundant proteins yield low sequence coverage (assuming that digestion is complete, the protein has a good digestion pattern, and the peptides do not suffer from unusually low ionization efficiencies). The protein score is derived from the individual ion scores and, using the same logic, the higher the protein score, the higher the abundance of a particular protein in the sample. As the gel was sliced into equal amounts and the protein score (Mowse) is loosely correlated to protein abundance in the sample, a plot of the gel slice versus Mowse score for a single protein can be generated. The purpose of this experiment was to plot what specific protein contributes to the visual band. Figure 5 shows the gel slice plotted against the Mowse scores for lactoferrin and serum albumin (obtained from the protein identifications from the 10% gel). The Schirmer method has a relatively low presence of lactoferrin in the regions marked 1 and 2 whereas the capillary has a high presence of lactoferrin in the same region. The opposite is true of serum albumin where it is observed a higher presence of serum albumin associated with the Schirmer method than the capillary collected tears.


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)

1D-SDS–PAGE of coomassie stained lactoferrin and serum albumin. Shown is a graph of gel slice versus Mowse score of lactoferrin and serum albumin to determine what protein is the dominating factor from the gel band.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: 1D-SDS–PAGE of coomassie stained lactoferrin and serum albumin. Shown is a graph of gel slice versus Mowse score of lactoferrin and serum albumin to determine what protein is the dominating factor from the gel band.
Mentions: The sequence coverage observed in the mass spectrometry experiments reflects the amount of the protein. Highly abundant proteins yield high sequence coverage while low abundant proteins yield low sequence coverage (assuming that digestion is complete, the protein has a good digestion pattern, and the peptides do not suffer from unusually low ionization efficiencies). The protein score is derived from the individual ion scores and, using the same logic, the higher the protein score, the higher the abundance of a particular protein in the sample. As the gel was sliced into equal amounts and the protein score (Mowse) is loosely correlated to protein abundance in the sample, a plot of the gel slice versus Mowse score for a single protein can be generated. The purpose of this experiment was to plot what specific protein contributes to the visual band. Figure 5 shows the gel slice plotted against the Mowse scores for lactoferrin and serum albumin (obtained from the protein identifications from the 10% gel). The Schirmer method has a relatively low presence of lactoferrin in the regions marked 1 and 2 whereas the capillary has a high presence of lactoferrin in the same region. The opposite is true of serum albumin where it is observed a higher presence of serum albumin associated with the Schirmer method than the capillary collected tears.

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