\set{final}

\def\Author{Ablonczy}
\def\author{ablonczy}
\def\year{2000}
\def\vol{6}
\def\anum{15}
\def\pages{109-115}
\def\txt_title{Mass spectrometric analysis of rhodopsin from light damaged rats}
\def\txt_authors{Zs. Ablonczy, D. R. Knapp, R. Darrow, D. T. Organisciak, Rosalie K. Crouch}

\def\rcvd{23 March 2000}
\def\accept{24 May 2000}
\def\publ{27 June 2000}
\def\pdfsize{}
\def\PMID{}


\include{mvstyle.hsm}

\| External links

\| Internal defs

\def\Eotvos{E\ouml tv\ouml s}
\def\Kuhn{K\uuml hn}

\article{

\title{Mass spectrometric analysis of rhodopsin from light damaged rats}


\authors{\mailto{ablonczy@musc.edu}{Zs. Ablonczy},\sup{1,2}
\mailto{knappdr@musc.edu}{D. R. Knapp},\sup{1} R. Darrow,\sup{3}
\mailto{dto@wright.edu}{D. T. Organisciak},\sup{3}
\mailto{crouchrk@musc.edu}{Rosalie K. Crouch}\sup{1}}

\institutions{\sup{1}Medical University of South Carolina, Charleston,
SC; \sup{2}\Eotvos\ University, Budapest, Hungary; \sup{3}Petticrew
Research Laboratory, Wright State University, Dayton, OH}

\correspondence{Rosalie K. Crouch, Ph.D., Department of Ophthalmology,
Medical University of South Carolina, 707 SEI 167 Ashley Avenue,
Charleston, SC, 29425; Phone: (843) 792-3031; FAX: (843) 792-5110;
email: \mailto{crouchrk@musc.edu}{crouchrk@musc.edu}}

\abstract

\abs_purpose{It is well established that the retina is damaged by
intense visible light. Rhodopsin has been proposed to be involved in
this process. We therefore undertook to examine whether rhodopsin
isolated from light damaged animals is structurally altered at the
molecular level.}

\abs_methods{Dark reared and dim cyclic light reared 8 week old
Sprague-Dawley rats were exposed to intense visible light and sacrificed
immediately or 24 h after exposure together with unexposed control
animals reared under the same conditions. Rod outer segments were
isolated by sucrose gradient ultracentrifugation, their membranes
treated with urea, then washed with Tris buffer. The rhodopsin
preparations were then reduced, pyridylethylated, delipidated, and
cleaved with CNBr. Reversed phase HPLC was used to separate the
fragments, and the effluent was analyzed online with a Finnigan LCQ ion
trap mass spectrometer. C-terminal phosphorylation was investigated
following Asp-N cleavage. MALDI-TOF mass spectrometry was used for the
identification of glycosylation.}

\abs_results{The rat rhodopsin protein was mapped with the exception of
two single amino acid fragments. The reported sequence was confirmed
with the exception of the controversial T/S320 residue, which was found
to be a threonine. Mono-, di-, tri-, and tetraphosphorylated forms of
rhodopsin were found in the light damaged animals. Three sites of
phosphorylation were confirmed with MS/MS (tandem mass spectral) data.
Single or double phosphorylations were found among these three sites, in
various combinations. Dark adaptation completely reversed the
phosphorylation in all light damaged animals. Other posttranslational
modifications were as previously reported.}

\abs_conclusions{Our results indicate that intense visible light
exposure of rats does not lead to oxidative or other primary structural
alterations in the rhodopsin protein of rod outer segments. We also
report that the mutated rhodopsin (P23H) is present in rat rod outer
segments from heterozygous animals and that residue 320 in both normal
and mutated rhodopsins is threonine, not serine.}

\introduction

\p{In 1966, Noell et al. first described visible light induced retinal
degeneration in rats [1]. His study and subsequent work indicate that
the action spectrum of this "light damage" is that of the absorption
spectrum of rhodopsin [1,2]. It is now recognized that light damage is
species-specific and is dependent on the duration and intensity of the
damaging light, the nature of the absorbing chromophores in the retina
[1,3-7], and the age and environmental history of the animals [8-11] as
well as their genetic characteristics [12,13]. There is compelling
evidence that extensive bleaching of rhodopsin induces oxidative stress
in the retina that can affect the entire cell. Long term exposure may
lead to lipid peroxidation of rod outer segments [14], but morphologic
changes manifest in the inner segment as well [15]. Oxidative and
apoptotic light induced fragmentation of DNA has been reported [16-19],
processes that are reduced by pretreatment of the animals with
antioxidants [20-24]. It is not clear, however, if the bleaching of
rhodopsin that initiates this damage has any oxidative, truncative, or
other effects on the rhodopsin protein itself. The purpose of this study
was to examine, by mass spectrometric methods, the structure of
rhodopsin from control and light damaged animals.}

\p{Over the past several years, mass spectrometry has been increasingly
applied to studies of protein structure [25-27]. Most commonly, mass
spectrometric methods have been used with soluble proteins that
generally yield soluble and chromatographically well-behaved peptide
cleavage products. Integral membrane proteins have posed serious
challenges for analysis by mass spectrometric methods, but methodology
has now been developed that allows observation of the entire sequence of
rhodopsin [28]. In the work reported here, we applied this method to the
study of rhodopsin derived from rod outer segments of light damaged
rats.}

\p{Light-stimulated phosphorylation of rhodopsin was first described 28
years ago [29]. Upon irradiation, rhodopsin is converted to an activated
state. The deactivation process involves phosphorylation of the protein
with rhodopsin kinase [30], and phosphorylation promotes arrestin
binding, which prevents the further activation of the visual cascade. In
vitro, it has been demonstrated that bleached rhodopsin can incorporate
up to nine moles of phosphate per mole of protein [31]. The most
probable phosphorylation sites are the hydroxylamino acids that lie in
the C terminal region of the protein [32-34]. Previous in vitro mass
spectrometric experiments identified single sites of rhodopsin
phosphorylation [35,36] while in vivo studies have shown multiple
phosphorylation sites on the protein [37,38]. In this study, we
identified three major sites of phosphorylation on the rhodopsin of
light damaged rats.}

\methods

\subsection{Animal care and rhodopsin extraction}

\p{Normal albino Sprague-Dawley (SD) rats, Royal College of Surgeons
(RCS) dystrophic rats (Harlan Inc., Indianapolis, IN) and heterozygous
rats having a P23H mutation in rhodopsin (line 3, from Dr. M.
LaVail, UCSF School of Medicine, San Francisco, CA) were reared in darkness or
in a weak cyclic light environment consisting of 12 h of 20-40 lux light
per day. The RCS dystrophic animals were 24 days of age, the P23H
mutated heterozygotes were 38-50 days, and the normal SD rats 60-75 days
when sacrificed. Animals were exposed to intense visible light for
various periods of time with unexposed controls being kept under normal
conditions. The animals reared in cyclic light were dark adapted for 16
h before sacrifice or light exposure. The length of exposure for P23H
mutated rats was 4 h for the cyclic and 2 h for the dark reared animal.
The exposure time for normal SD rats was 8-24 h. Only non-light exposed
RCS rats ("control RCS rats") were used in these experiments. The
chambers used for light exposure and the exposure conditions have
previously been described [39]. Following light exposure some animals
were kept in darkness for 24 h while others were sacrificed immediately.
All rats were sacrificed in red light in carbon dioxide saturated
chambers and their retinas excised within 2 min of death. Rod outer
segments were immediately isolated by sucrose gradient
ultracentrifugation [24] and rhodopsin prepared by the method of
McDowell and \Kuhn\ [40]. The final purified sample, from 8-12 pooled
retinae, contained approximately 175 \mu g of protein. Care was taken to
purge all buffers with nitrogen and to store the extracted protein under
argon to avoid oxidation. The samples were then wrapped in foil, and
kept in the dark at -80 \deg C, then shipped on dry ice from the Wright
State University to the Medical University of South Carolina for further
analysis. The use of animals in this investigation conformed to the ARVO
statement for the use of animals in research.}

\subsection{Generation of peptides for sequence analysis}

\p{The preparation of rhodopsin samples for mapping purposes followed
the methods developed earlier for the mass spectrometric analysis of
integral membrane proteins [28]. The purified rhodopsin samples were
reduced with tributylphosphine, (Aldrich Chem. Co., Milwaukee, WI),
alkylated with 4-vinylpyridine (Sigma Chem. Co., St. Louis, MO), and
delipidated by precipitation in ethanol. The delipidated protein was
cleaved with CNBr and then evaporated to dryness under vacuum. The dried
fragment mixture was dissolved in 5 mL of solution equivalent to the
initial HPLC gradient mobile phase (97.5% A and 2.5% B), where A is
0.05% aqueous TFA solvent and B is 2:1 isopropanol/acetonitrile
containing 0.05% TFA. The sample was then loaded onto a 2.1 mm x 100 mm
C4 Aquapore column (Perkin Elmer Brownlee Column, Bodman Industries,
Aston, PA) at 400 \mu L/min of 2.5% B for 12.5 min. The peptides were
eluted at a flow of 400 \mu L/min with a gradient of 2.5-60% B in 60 min
and 60-97.5% B in 20 min. The HPLC was done by an HP1100 series HPLC
instrument (Agilent Technologies, Palo Alto, CA). The C terminal
fragment was dried under vacuum, dissolved in 50 \mu L of 9:1 100 mM
ammonium bicarbonate at pH 8.3/acetonitrile and digested with 0.5 \mu g
trypsin in 1 mM HCl overnight at 37 \deg C. The cleavage mixture was
dried under vacuum and dissolved to yield 20 \mu L of solution
equivalent to the initial HPLC gradient mobile phase (97.5% A and 2.5%
B), where A is 0.1 M acetic acid and B is acetonitrile. The 20 \mu L
sample was loaded onto a 0.32 mm x 150 mm C4 Intersil (Micro-Tech
Scientific, Sunnyvale, CA) capillary column (at 5 \mu L/min of 2.5% B
for 5 min) and the peptides eluted with a gradient of 2.5-97.5% B in 60
min.}

\p{For the determination of the amount and location of C terminal
phosphorylation, samples were centrifuged for 15 min at 100,000 g, the
supernatant was removed and the pellet suspended in 100 \mu L 10 mM Tris
at pH 7.5. The protein was cleaved overnight at 37 \deg C with 50 ng
endoproteinase Asp-N (Sigma Chemical Co., St. Louis, MO) at
approximately 1/2000 enzyme to substrate ratio, dissolved in water. After
centrifugation (15 min at 100,000 g), the supernatant was dried under
vacuum and dissolved to yield 5 mL in the initial HPLC gradient mobile
phase (97.5% A and 2.5% B), where A is 0.05% aqueous TFA solvent and B
is acetonitrile containing 0.05% TFA. The 5 mL sample was loaded onto a
2.1 mm x 100 mm C4 Aquapore column (at 400 \mu L/min of 2.5% B for 12.5
min). The peptides were eluted at a flow of 200 \mu L/min with a
gradient of 2.5-5% B in 5 min, 5-15% B in 10 or 20 min.}

\p{The glycosylated rhodopsin fragment was dried under vacuum, dissolved
in 50 mM ammonium bicarbonate at pH 7.6 and treated for 30 min at 25
\deg C with N-glycosidase F and analyzed on MALDI-TOF (matrix assisted
laser desorption ionization-time of flight mass spectrometry)
instrument with the procedure below.}

\subsection{Mass spectrometry}

\p{The column effluent from the 2.1 mm HPLC columns was split and 10% of
the flow was directed into the ESI source of a Finnigan LCQ ion trap
mass spectrometer (Thermo Instrument Systems Inc., Waltham, MA).
Capillary column effluents were not split. Data were acquired using LCQ
version 1.2 software, details previously described [28]. MS (mass
spectral) data were acquired by repetitive scanning with MS/MS data
automatically acquired for the most intense precursor ion in each MS
spectrum. The remaining 90% of the effluent was collected in 2 min
fractions, dried under vacuum and stored for later use. These fractions
were further analyzed with a Perseptive Voyager DE MALDI-TOF (delayed
extraction MALDI-TOF) instrument (PE Biosystems, Foster City, CA) using
\alpha-cyano-4-hydroxycinnamic acid as matrix. The
\alpha-cyano-4-hydroxycinnamic acid was dissolved in 70% acetonitrile.
The dried HPLC fractions were each dissolved in 4 \mu L of 70%
acetonitrile. A 0.3 \mu L aliquot of the solution was spotted on the
MALDI plate followed by 0.9 \mu L of the matrix solution. The spots were
allowed to dry on the plate together with external standards for the
calibration of the instrument.}

\results

\p{The rat rhodopsin samples were analyzed with two mass spectrometric
techniques, ESI-MS (electronspray ionization-mass spectrometry) and
MALDI-MS. In ESI-MS, the effluent of an HPLC column passes through a
needle tip at high electrical potential, which results in a fine mist of
highly charged droplets. After the evaporation of the solvents this
yields multiply-charged peptide ions (with several different charge
states), detected by the mass analyzer. The ion trap instrument is
capable of storing a selected ion and fragmenting it by collision with
inert gas. The fragmentation occurs mainly at peptide bonds. By
detecting the masses of the different fragments, the amino acid sequence
of 20-30 amino acid peptides can easily be reconstituted (MS/MS
spectra). MALDI-MS requires mixing of the solution with an excess of
small matrix molecules that absorb laser light. Upon excitation, the
matrix molecules explode from the sample plate, ionizing the peptides in
the solution. These peptides are then sorted by their time of flight in
a vacuum tube by their mass/charge ratio and their abundance is
detected. The MALDI technique resembles a protein gel but it is much
more accurate.}

\p{Using these techniques, the rhodopsin from Sprague-Dawley rats was
completely mapped with the exception of the two single amino acid
fragments. \figref{1} shows the amino acid sequence and a
two-dimensional model of rat rhodopsin, and \tabref{1} indicates its
CNBr fragments.}

\p{MS/MS data were collected for 15 of the total 18 CNBr fragments. The
two single amino acid fragments are not detectable with the instrument
and no sequence data were collected for the glycosylated fragment
2. However fragment 2 was identifiable in the base peak
chromatogram. The difference between the masses of fragment 2
from wild type rats and the P23H transgenics was also observed (+40 Da).
In the case of P23H both mass peaks are present because of the two
rhodopsins being present in the heterozygotes. MS/MS data were not
obtained for fragment 2 due to the length of the peptide chain
and the attached heterogeneous oligossacharides. By collecting 2 min
fractions from the HPLC effluent, it was possible to find the exact
location of fragment 2 in the gradient. These fractions were
deglycosylated with N-glycosidase F and the peptide identified by the
loss of 1096 Da, the mass for GlcNAc(Man)\sub{3}(GlcNAc)\sub{2}, the most
abundant glycoform obtained by mass spectrometry earlier [41] (data not
shown). \figref{2} shows LCQ base peak chromatograms for dark reared,
light damaged animals and a cyclic reared control preparation, with the
most abundant CNBr fragments indicated (fragments 3, 4, 5, 6, 7, 8, 14,
15, and 18). The fragments not indicated (fragments 2, 9, 10, 11, 12, 13,
and 17) were found in the background. The base peak chromatogram shows
the amount of the most abundant detected precursor ion eluting from the
HPLC column at each time point. Since the detection limit was 400-2000 Da,
only fragments 3 and 7 were detected as +1 charge state
ions, the other fragments were detected in one of their higher charge
states (see \tabref{1} for reference). The protein sample was not
completely clean and there were contaminants also in the HPLC gradient
(the most prominent one had a mass of 437 Da). As it can be seen in
\figref{2}, some of the fragments contained were oxidized tryptophans
due to aged CNBr. The peaks are normalized to the most abundant ion in
each chromatogram and therefore their relative amounts are not
comparable.}

\p{The C-terminal fragment is also relatively large and has extensive
posttranslational modifications (palmitylation and possible
phosphorylation). The MS/MS data for this fragment were sufficient for
the identification of the fragment but gave no information on the
location of the modifications. Therefore, the collected fragments were
digested with trypsin. The trypsin digest of fragment 18 provides three
smaller peptide fragments which were easier to analyze. The MS/MS data
for the first trypsin fragment of the C-terminal CNBr fragment 18 showed
the amino acid at position 320 to be threonine (\figref{3}). The amino
acid is identified by the 101 Da mass difference between the b\sub{2} and
b\sub{3} or y\sub{5} and y\sub{6} fragment ions. There is also a
posttranslational modification on the same fragment. The loss of 238 Da
from y\sub{7} and some other ions indicated palmityl group loss, and the
difference of 341 Da between y\sub{3} and y\sub{4} shows, that cysteine
322 was palmitylated. However, the mass ladder implies, that cysteine
323 was also palmitylated.}

\p{Apart from the above mentioned four CNBr fragments, the MS/MS data
collected during a single HPLC run were sufficiently complete not only
for the identification of the fragments but for the possible
posttranslational modifications as well. The posttranslational
modifications were found to be as previously reported for bovine
rhodopsin. The first amino acid is acetylated, asparagines 2 and 15 are
glycosylated, cysteines 322 and 323 are palmitylated, and, in the case
of light adapted samples, there was phosphorylation on the C terminus.
\figref{1} shows the sequence as well as the posttranslational
modifications on the protein. No sequence modification between the light
damaged and control animals was found. For the P23H samples, the
appropriate mass shift was observed in fragment 2.}

\p{When light damaged animals were not dark-adapted before sacrifice,
phosphorylation was observed. Phosphorylation was identified by a mass
shift of +80 Da for HPO\sub{3} (or its appropriate multiple for multiple
phosphorylation) on the observed fragments. In order to identify the
sites of phosphorylation, Asp-N digestion was used to cleave the last 19
amino acids of the protein [35,38]. Three main sites of phosphorylation
were identified by MS/MS data. Threonine 336 as well as serines 334 and
338 were found to be monophosphorylated and their various combinations
could be identified as the di- and triphosphorylated states.
Tetraphosphorylation was also present, but the sites were not
identified. Di- and triphosphorylation MS/MS data are difficult to
analyze because of the low intensity of the mass peaks. There was an
indication of possible phosphorylation on S 343 or T 342 on the doubly
and triply phosphorylated species. T 340 was not found to be
phosphorylated. \figref{4} shows comparison of the phosphorylation
states of dim cyclic light reared light damaged/no dark-adaptation,
light damaged/dark-adapted and control/dark-adapted animals. As can be
seen, that light damaged and not dark-adapted samples contained multiple
phosphorylation (identified by the +40 Da mass shift over the
(M+2H)\sup{+2} ion). The base peak chromatogram shows only
triphosphorylation, but the mass spectrum reveals the very small amount
of tetraphosphorylation. As seen by the other samples in the middle and
on the bottom, dark-adaptation completely reversed the phosphorylation
of the protein, even in the light damaged animals. It can also be seen
that there is a very small peak (980 Da) in the control/dark adapted
spectrum that indicates a small amount of phosphorylation.}

\p{Transgenic rats having a histidine substituted at the proline 23
position are known to be extremely light sensitive [42,43] and this
mutation has been found in patients with one form of autosomal dominant
retinitis pigmentosa [44]. We therefore examined the rhodopsin isolated
from control and light exposed P23H transgenic rats. We did find the
mutation at position 23 in the rod outer segment rhodopsin. However, we
found no evidence for other alterations of either the mutant rhodopsin
or the native rhodopsin from these animals.}

\p{Although the animals used for controls were SD rats, rhodopsin from
RCS rats, not light damaged, was also examined and found to have
identical sequence as the other wild type SD rats.}

\discussion

\p{The mass spectrometric analysis protocol developed for the mapping of
integral membrane proteins and successfully applied previously to
bacteriorhodopsin (BR) and bovine rhodopsin [28] proved equally
applicable to rat rhodopsin. In spite of the small amount of protein
(the samples contained approximately 175 \mu g or 5 nM of protein), we
observed 90% of the entire sequence in a single experiment. The same
posttranslational modifications were found in rat rhodopsin as had been
reported for bovine rhodopsin [28]. Glycosylation was also found to be
present with the same most abundant glycoform [28]. For rat rhodopsin,
the Swiss-Prot gene sequence data reported a serine at 320 [45] as well
as a possible threonine at this site (data not published). Our results
show that rat rhodopsin contains a threonine at residue 320.}

\p{Our goal was to explore whether rat rhodopsin protein is damaged by
extensive in vivo light exposure. Our results do not show evidence for
any alterations or modifications in the primary sequence of the protein.
Transgenic animals having a P23H mutation in rhodopsin are known to be
extremely sensitive to light damage [42,43]. No light induced
alterations in rhodopsin were found for these animals as well. This
result was confirmed with the mass peaks of the base peak chromatograms
and by sequencing individual peptide fragments. However, we did find
unequivocal evidence that the mutated rhodopsin, as well as normal
rhodopsin, is present in the rod outer segments from these animals. All
rhodopsin fragments, except for the two single amino acid fragments and
the glycosylated N terminal fragment were completely sequenced. The loss
of masses corresponding to palmitates was also observed, strong evidence
for the palmitylated state of the C terminal CNBr fragment.}

\p{It is well known that the C terminal region of light adapted
rhodopsin is phosphorylated. Our results show extensive phosphorylation
on the C terminal serines and threonines of rat rhodopsin. Previous in
vivo experiments on mice that had received light flashes or continuous
irradiation showed monophosphorylation at serine 338 and 334
respectively [37]. Hurley et al. reported multiple phosphorylations
(mono to tri) of mouse rhodopsin isolated from intact retinae exposed to
light in vitro [38]. In the experiments reported here, the rhodopsin was
phosphorylated in vivo. The MS/MS data showed three main
monophosphorylation sites and various diphosphorylation combinations at
these sites. Triphosphorylation was also observed in various
combinations of the three main monophosphorylation sites. We also
obtained a very small intensity tetraphosphorylated peak based upon
molecular weight measurement, but it was not confirmed by sequence
analysis. This result agrees with the previous finding that extensive
rhodopsin bleach causes multiple phosphorylations [38]. However, the
extensive in vivo light damaging bleach, which leads to visual cell
death, does not result in phosphorylation of \gt 40% of the total amount
of rhodopsin. Since the animals were sacrificed and the rhodopsin
excised immediately after light damage, phosphatase activity should not
be a major factor. However, the action of phosphatases in causing
dephosphorylation cannot be ruled out. It was reported earlier that
monophosphorylation may be present in fully dark-adapted retinas [38],
and our results confirm that there may be a very small amount of
rhodopsin phosphorylation (3-4%) in dark-adapted light damaged or
control retinas. The MS/MS data show that the site of phosphorylation
can be on any one of the three main monophosphorylation sites.}

\p{In conclusion, we have mapped rhodopsin peptides from control and
light damaged rats by mass spectrometry and have confirmed that the
amino acid at position 320 is threonine. Our results do not show any
alterations in rhodopsin following exposure to levels of light that
cause retinal damage, aside from the identification of
multiphosphorylated states. However, after dark adaptation, no
differences between the experimental and control groups were observed.
Even using the light sensitive P23H transgenic animals, no modifications
of the protein were observed. It cannot be ruled out that there is a
form of the protein that is highly modified and therefore is not
isolated through our sucrose flotation procedures, not cleaved in our
fragmentation of the protein for mass spectrometry, or not eluted from
our HPLC separation of the peptides. However, as the amount of protein
isolated from the light damaged animals and the controls was equivalent
and as the concentrations of the peptides appeared comparable, we
conclude that the majority of the protein has not been altered. Our data
therefore indicate that primary structural changes in rhodopsin are not
a consequence of the well-known light damage effect that disrupts the
organization of retinal photoreceptor cells.}

\acknowledgements

\p{We thank Drs. K. Schey and Jim Hurley for helpful discussions. Dr. M.
LaVail generously supplied the P23H transgenic rats. Funding was
provided by NIH grants EY-01959, EY-04939, and EY-08239; a private
endowment to Dr. Organisciak from M. Petticrew and an unrestricted grant
to MUSC from the Foundation for Prevention of Blindness. The mass
spectrometry work was performed in the MUSC Mass Spectrometry
Institutional Research Resource Facility.}

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}

\beginfigures

\figfile{1}{
\figtitle{1}{Two-dimensional model and amino acid sequence of rat
rhodopsin}

\p{The first 39 amino acids (dashed green circles, green and black
letters) were not sequenced with MS/MS. The blue dashed circles and blue
letters for the last 19 amino acids denote the fragment cleaved by
Asp-N. The detected posttranslational modifications are also indicated
as well as the site of the P23H mutation (black letter). The
posttranslational modifications are identical with those in bovine
rhodopsin. Notice that amino acid 320 is a threonine and not a serine
(black letter). Phosphorylation sites are shown in black. The numbers
indicate the beginning of the corresponding CNBr fragment.}

\ctr{\jpgimage{1}{575}{431}{77}}

}

\figfile{2}{
\figtitle{2}{LCQ base peak chromatograms of rat rhodopsin}

\p{The chromatograms of dark reared light damaged rhodopsin (top) and
the cyclic dim light reared control rhodopsin (bottom) are shown. The
most abundant CNBr fragments are indicated. Other fragments can also be
found in the background. Additional peaks are contaminants. The
retention time (numbers on top) and the mass of the most intense
precursor ions (numbers below) are shown. There were no sequence
differences or other modification observed between control and light
damaged, dark and cyclic dim light reared animals.}

\ctr{\jpgimage{2}{480}{360}{33}}

}

\figfile{3}{
\figtitle{3}{Sequence conflict in rat rhodopsin}

\p{The figure shows the MS/MS sequence of the first trypsin fragment of CNBr
treated rhodopsin fragment 18. The observed mass of this fragment was different
from the mass expected from the gene sequence. The sequence confirms that this
difference is due to amino acid 320 (the second threonine in the figure)
being a threonine not a serine. In the mass spectrum, PA stands for
palmityl group. X stands for palmityl cysteine in the sequence. Notice
the loss of the palmityl group from the palmityl cysteines. Underlining
indicates the observed masses.}

\ctr{\jpgimage{3}{671}{503}{53}}

}

\figfile{4}{
\figtitle{4}{Light damage phosphorylation of rat rhodopsin}

\p{Base peak chromatograms (left) and mass spectra (right) from Asp-N
cleaved C terminal fragments of rhodopsin are shown. The sample shown at
the top was extracted from cyclic dim light reared light damaged and not
dark-adapted animals. The one in the middle is cyclic dim light reared
control, and the one below is cyclic dim light reared light damaged and
24 h dark-adapted. Notice the amount of phosphorylation on the first
sample. Three sites of phosphorylation are confirmed with MS/MS data
(not shown). No sequence information was obtained for the
tetraphosphorylated species. There were multiple mono- and
diphosphorylation species. Notice that dark adaptation completely
reverses phosphorylation. The difference between the time scale and the
sensitivity of the modified and unmodified samples was due to
differences in experimental conditions.}

\ctr{\jpgimage{4}{671}{503}{68}}

}

\begintables

\tabfile{1}{
\tabtitle{1}{The CNBr fragmentation of rat rhodopsin}

\ctr{\box{\gifimage{1}{475}{396}{15}}}

\p{\sup{a}The first acetylated methionine was not detected with LC/MS/MS.\br
\sup{b}Observed with MALDI as an incomplete cleavage after the first methionine.\br
\sup{c}No sequence confirmation with LC/MS/MS but observed with MALDI-TOF.\br
\sup{d}The most abundant glycoform.\br
\sup{e}Observed with the incorporated tryptophan oxidized due to aged CNBr.\br
\sup{f}Also observed with homoserine C terminal ending.\br
\sup{g}Not detected.\br
\sup{h}Also observed with HPO\sub{3} addition in light damaged samples.}

}