Acceleration of age-induced proteolysis in the guinea pig lens nucleus by in vivo exposure to hyperbaric oxygen: A mass spectrometry analysis

Abstract

Hyperbaric oxygen (HBO) treatment of animals or ocular lenses in culture recapitulates many molecular changes observed in human age-related nuclear cataract. The guinea pig HBO model has been one of the best examples of such treatment leading to dose-dependent development of lens nuclear opacities. In this study, complimentary mass spectrometry methods were employed to examine protein truncation after HBO treatment of aged guinea pigs. Quantitative liquid chromatography-mass spectrometry (LC-MS) analysis of the membrane fraction of guinea pig lenses showed statistically significant increases in aquaporin-0 (AQP0) C-terminal truncation, consistent with previous reports of accelerated loss of membrane and cytoskeletal proteins. In addition, imaging mass spectrometry (IMS) analysis spatially mapped the acceleration of age-related αA-crystallin truncation in the lens nucleus. The truncation sites in αA-crystallin closely match those observed in human lenses with age. Taken together, our results suggest that HBO accelerates the normal lens aging process and leads to nuclear cataract.

Keywords: Alpha crystallin; Aquaporin-0; Hyperbaric oxygen; Lens; Mass spectrometry; Nuclear cataract; Protein truncation; αA66-80 peptide.

Figure 1:. LC-MS analysis of guinea pig lens AQP0.

The base peak chromatogram of a lens tryptic digest of the membrane fraction from a control 25-month-old guinea pig lens cortex is shown in the top panel where m/z values of ions observed in the the major base peaks are labeled. Selected ion chromatograms (SICs) for expected N- and C-terminal tryptic peptides are shown in the lower panels. Each SIC is accompanied by the AQP0 residue numbers of the selected peptide, the observed ion charge state, the m/z of the plotted signal, and the peptide sequence. Asterisks indicate the peak of interest. Bold N in the peptide sequence indicates Asp due to partial or complete deamidation.

Figure 2:. LC-MS analysis of C-terminal truncation products of guinea pig lens AQP0.

Selected ion chromatograms for truncated C-terminal tryptic peptides in the cortex (A) and nucleus (B) regions of an 81x HBO-treated lens from a 25 month old animal (sequences are indicated next to the eluted peaks). Asterisks indicate the peaks of interest. Bold N in the peptide sequence indicates partial or complete deamidation.

Figure 3:. Quantitative analysis of AQP0 C-terminal truncation in control and HBO treated guinea pig lenses.

A: Samples from cortex; B: samples from nucleus (note the different vertical axis scales in the two figures). Percent truncation at 5 different C-terminal amino acid residues is shown. Animals were treated with HBO either 81 or 84 times over a 7-month period (25-months at time of analysis). The y-axis indicates the ratio of the truncated peptide signal to the corresponding non-truncated peptide signal of AQP0 tryptic peptide containing residues 239–259. Results are expressed as means +/− S.D. n=4 * indicates a statistically significant difference (p<0.05).

Figure 4:. Imaging mass spectrometry of lens crystallin peptides in guinea pigs treated with HBO.

Spatial distribution of selected IMS signals that represent crystallin truncation peptides. Sequence assignments are shown. The ages of the animals were 22 months for HBO-treated, and either 2 or 22 months for untreated controls. Experimental animals were treated 43 times with HBO over a 15-week period. The results shown are representative of two experiments.

Figure 5:. Imaging mass spectrometry of lens crystallin peptides in guinea pigs treated with HBO over time.

Spatial distribution of selected IMS signals in lenses as a function of 15X, 30X, or 43X treatments with HBO (19, 20.5 and 22 months of age, respectively). Sequence assignments are shown.