2. Add 100 μL of chloroform and vortex.
3. Add 300 μL of Milli-Q water—mixture will become cloudy
with precipitate—and vortex.
4. Centrifuge for 1 min at 14,000 Â g. The result is three layers: a
sizeable aqueous layer on top, a circular flake of protein in the
interphase, and a smaller chloroform layer at the bottom.
5. Remove the top aqueous layer carefully, trying not to disturb
the protein flake.
6. Add 400 μL of methanol and vortex.
7. Centrifuge for 5 min at 20,000 Â g.
8. Remove as much methanol as possible. Be careful, because the
pellet is delicate. You should be able to remove all but a few μL
of methanol with care, which will speed drying.
9. Allow it to air-dry.
10. The pellet is resuspended in Milli-Q water and submit to an
in-gel concentration and in-gel digestion (see next section).
Tear Samples
Tear fluid is a complex mixture of proteins, lipids, salts, and other
organic molecules produced by the lacrimal glands. Usually, the
protein concentration is 5–7 mg/mL [39]. The tear function is the
lubrication of the eye, delivery of nutrients, and maintenance of the
refractivity of the cornea [40]. Moreover, tear provides an effective
chemical barrier on the surface of the eye by secreting antimicrobial
and immunomodulatory proteins (AMP) that protect against infections [41]. Currently, there have been identified more than 1500
tear proteins by different proteomics techniques; for more details,
see Cso ˝sz et al. [29]. Major tear proteins are lactotransferrin,
lysozyme-C, prolactin-inducible protein, lacritin, etc., that are
involved in the defense against pathogens [41]. Other tear proteins
are produced by the epithelial cells, such as dermcidin, defensins,
etc., and others are filtered from the blood, such as albumin [42].
The tear proteome profiling study is considered a challenge
because only a small volume of tear fluid (<5 μL) can be collected
in a clinical laboratory under normal operational conditions. That
being so, only a few proteins were identified by LC-MS/MS techniques [28, 42].
For proteomic analysis, tear samples can be obtained from
microcapillary tubes or Schirmer tear test strips [43–45].
Tear Sample Preparation
1. Tear proteins are collected by the capillary method: in this
method, a maximum of 15 μL of tears is collected and stored
immediately at À80
C until their use.
(a) 10 μL of tears (ca. 50 μg of protein) is precipitated in
order to eliminate lipids, salts, and other organic molecules present in these samples. For this purpose, the acetone method precipitation is used.
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3. Add 300 μL of Milli-Q water—mixture will become cloudy
with precipitate—and vortex.
4. Centrifuge for 1 min at 14,000 Â g. The result is three layers: a
sizeable aqueous layer on top, a circular flake of protein in the
interphase, and a smaller chloroform layer at the bottom.
5. Remove the top aqueous layer carefully, trying not to disturb
the protein flake.
6. Add 400 μL of methanol and vortex.
7. Centrifuge for 5 min at 20,000 Â g.
8. Remove as much methanol as possible. Be careful, because the
pellet is delicate. You should be able to remove all but a few μL
of methanol with care, which will speed drying.
9. Allow it to air-dry.
10. The pellet is resuspended in Milli-Q water and submit to an
in-gel concentration and in-gel digestion (see next section).
Tear Samples
Tear fluid is a complex mixture of proteins, lipids, salts, and other
organic molecules produced by the lacrimal glands. Usually, the
protein concentration is 5–7 mg/mL [39]. The tear function is the
lubrication of the eye, delivery of nutrients, and maintenance of the
refractivity of the cornea [40]. Moreover, tear provides an effective
chemical barrier on the surface of the eye by secreting antimicrobial
and immunomodulatory proteins (AMP) that protect against infections [41]. Currently, there have been identified more than 1500
tear proteins by different proteomics techniques; for more details,
see Cso ˝sz et al. [29]. Major tear proteins are lactotransferrin,
lysozyme-C, prolactin-inducible protein, lacritin, etc., that are
involved in the defense against pathogens [41]. Other tear proteins
are produced by the epithelial cells, such as dermcidin, defensins,
etc., and others are filtered from the blood, such as albumin [42].
The tear proteome profiling study is considered a challenge
because only a small volume of tear fluid (<5 μL) can be collected
in a clinical laboratory under normal operational conditions. That
being so, only a few proteins were identified by LC-MS/MS techniques [28, 42].
For proteomic analysis, tear samples can be obtained from
microcapillary tubes or Schirmer tear test strips [43–45].
Tear Sample Preparation
1. Tear proteins are collected by the capillary method: in this
method, a maximum of 15 μL of tears is collected and stored
immediately at À80
C until their use.
(a) 10 μL of tears (ca. 50 μg of protein) is precipitated in
order to eliminate lipids, salts, and other organic molecules present in these samples. For this purpose, the acetone method precipitation is used.
116
Maria del Pilar Chantada-Va ´ zquez et al.