\set{final} \def\Author{Robinson} \def\author{robinson} \def\vol{12} \def\year{2006} \def\anum{79} \def\pages{704-711} \def\txt_title{Quantitative measurement of young human eye lens crystallins by direct injection Fourier transform ion cyclotron resonance mass spectrometry} \def\txt_authors{Noah E. Robinson, Kirsten J. Lampi, J. Paul Speir, Gary Kruppa, Michael Easterling, Arthur B. Robinson} \def\rcvd{31 March 2006} \def\accept{20 June 2006} \def\publ{21 June 2006} \def\pdfsize{} \def\PMID{} \include{mvstyle.hsm} \| External links \| Internal defs \article{ \title{Quantitative measurement of young human eye lens crystallins by direct injection Fourier transform ion cyclotron resonance mass spectrometry} \authors{\mailto{noah@oism.org}{Noah E. Robinson},\sup{1} Kirsten J. Lampi,\sup{2} J. Paul Speir,\sup{3} Gary Kruppa,\sup{3} Michael Easterling,\sup{3} \mailto{art@oism.org}{Arthur B. Robinson}\sup{1}} \institutions{\sup{1}Oregon Institute of Science and Medicine, Cave Junction, OR; \sup{2}Oregon Health and Science University, Portland, OR; \sup{3}Bruker Daltonics Corporation, Billerica, MA} \correspondence{Noah E. Robinson, Oregon Institute of Science and Medicine, 2251 Dick George Road, Cave Junction, OR, 97523; Phone: (541) 592-4142; FAX: (541) 592-2597; email: noah@oism.org} \abstract \abs_purpose{Human eye lenses at birth are primarily constructed of 12 distinct crystallins and two truncated crystallins. The molecular weights of these 14 proteins vary between about 20,000 and 30,000 Da. The relative amounts of these molecules and their post-synthetic changes with age are of substantial interest in the study of lens biochemistry and lens pathology. Fourier transform mass spectrometry of unfractionated lens homogenates now permits precise quantitative measurement of the relative amounts of lens crystallins. We report herein the measurement of the 14 crystallins in 10 pairs of lenses from humans between the ages of 2 and 300 days.} \abs_methods{Eye lenses were obtained from human donors of various ages in the first year of life. These lenses were homogenized in 0.02 M phosphate buffer at pH 7.0 with 0.001 M EDTA, desalted by washing over a 3,000 Da filter, and injected directly into the nanospray source of a hybrid Fourier transform ion cyclotron resonance mass spectrometer, Qq-FT(ICR)MS, equipped with a 12 Tesla magnet. The crystallins were quantitatively ionized and mass analyzed in the ICR cell of the mass spectrometer. The detected signals of all of the isotopic and charge state species for each crystallin were normalized and summed to determine the protein quantities.} \abs_results{The relative amounts of the 14 crystallins are found to be quite similar from individual to individual at birth. These amounts are in integer ratios to one another that suggest important structural relations within the lens. In two cases, the relative amounts of \alpha A- and \beta B2-crystallin change proportionally to the logarithm of age during the first year, with \alpha A- decreasing and \beta B2-crystallin increasing. The changes in \alpha A- and \beta B2-crystallin are mutually offsetting, with \alpha A-crystallin decreasing from 30% to 18% and \beta B2-increasing from 12% to 24%.} \abs_conclusions{These observations suggest that the human eye lens at birth is constructed of crystallins in which the numbers of crystallin molecules have regular integral relationships to each other. As the lens develops during the first year, some of these relationships change. While the functional significance of the reciprocal decrease in \alpha A- and increase in \beta B2-crystallin is not known, \beta B2-crystallin may substitute for \alpha A-crystallin in the lens structures synthesized during the year after birth. Direct injection FT(ICR)MS of unfractionated lens was found to be an excellent method for the quantitative measurement of lens crystallins.} \introduction \p{The lens of the human eye is constructed primarily of an ordered array of lens fiber cells [1]. The principal components within these cells are lens crystallins, of which there are 14 known types in the young, 12 primary gene products and two NH\sub{2}-terminally truncated products of two of the primary 12 [2,3]. These proteins are packed closely together at very high concentrations in such a way as to provide the lens with its unique optical properties. The abundances of the individual crystallins vary over a range of about 20 fold in the lenses of newborn children [2]. It is to be expected that the relative amounts of the different crystallin subunits correspond to their arrangement in the lens and reflect the specific lens supermolecular structures in which they participate.} \p{As the lens ages, the crystallins also age and change their properties [4-8]. Sometimes these changes in properties cause pathological alterations leading to lens opacity, as in cataract [9]. In order to understand these alterations and to understand the fundamental structure of the lens, it is of value to know the nature of the ordered associations of crystallin subunits and the changes that occur in these ordered arrays with age. This understanding has been impeded, however, by the lack of convenient and reliable means for quantitatively measuring the amounts of the individual crystallins.} \p{We report, herein, the development of a new method for quantitative measurement of lens crystallins and the application of this method to the measurement of the relative amounts of the 14 crystallins in human eye lenses at various ages during the first year of life.} \p{This method is based upon direct nanospray injection of lens homogenates into a 12 Tesla ion cyclotron resonance Fourier transform mass spectrometer. Each lens measurement requires about 20 min of mass spectrometer operation and provides simultaneous and remarkably reliable quantitative values for all 14 crystallins.} \methods \subsection{Preparation of lens crystallins} \p{Eye lenses from 10 human donors of ages 2 (4), 23, 25, 30, 90, 150, and 300 days were obtained from the Lions Eye Bank of Oregon and processed as previously described [6]. The lenses were homogenized in 1.0 ml of 0.02 M phosphate buffer at pH 7.0 with 0.001 M EDTA, centrifuged to remove any insoluble material, and desalted by repeated H\sub{2}O centrifugal washing over 3,000 Da (Millipore, Billerica, MA) filters. As a result of the young ages of the donors, the amount of insoluble material in these samples was negligible. Other investigators have similarly reported that, up to 1 year of life, 97% of the total human lens protein is soluble [10]. These eye lens protein solutions were diluted with 50:50:0.1 H\sub{2}O:acetonitrile:acetic acid for mass spectrometry. For lenses from donors older than studied here where insoluble lens crystallin fractions are present, a different nanospray solvent must be used.} \subsection{Fourier transform mass spectrometry} \p{The lens protein solutions were measured in a Bruker Daltonics 12.0 Tesla Apex Qe FTMS mass spectrometer manufactured by Bruker Daltonics, Inc. (Billerica, MA). Each sample was flow injected into a Bruker on-line nanospray source at a flow rate of 5 \mu l/h and a scan rate of 20 scans/min for a period of 20 min with all scans cumulatively summed during measurement and before Fourier transformation. The average resolution was about 200,000, where resolution=(mass)/(peak width at half height). The FTMS utilizes ion image current detection wherein coulomb forces between the circulating ions and the collector plates in the ICR cell result in an electric current flow between the plates. This current is directly proportional to the number of ions and to the charge on each ion.} \p{The superb linear correspondence between detector signal and number of ions present has been previously demonstrated [11]. Moreover, demonstrating this further, the samples measured herein varied in total protein concentrations over a more than 10 fold range, while the measured ratios of crystallins remained remarkably constant, as reported in \tabref{1}.} \subsection{Calculation of relative percentages of crystallins} \p{Fourier transformation of the collected data produces about 300 mass spectral peaks of significant intensity for each individual protein. \figref{1} illustrates these peaks for \beta B2-crystallin in a 2-day-old human lens sample. The \beta B2-crystallin molecules become charged to varying extents in the nanospray source. \figref{1}{A} shows the isotopic distribution for those with a charge of +22. These 20 isotopic peaks arise from the presence, at various positions in the protein, of elemental isotopes that have masses greater than \sup{1}H, \sup{12}C, \sup{14}N, \sup{16}O, and \sup{32}S. The peaks seen in the distribution in \figref{1}{A} are the sixth through the twenty-fifth peaks. Isotope peaks 1 through 5 are present in negligible quantities. \figref{1}{B} shows the distribution of charge states, each of which has an isotopic distribution. These states range from the least charged at +13 to the most charged at +30. \beta B2-crystallin has 20 significant isotopic peaks for each charge state. With 18 charge states, the total number of \beta B2-peaks in the mass spectrum is 360.} \p{The relative quantities in \figref{1}{B} are the summed areas of the peaks in the isotopic envelopes as shown in \figref{1}{A}, after correction for charge. Since the ion image current is proportional to charge, molecules that carry a greater charge produce a higher current. A linear correction has been made so that this difference is removed. These quantities have been normalized to 100 for the most abundant charge state. The distribution of charge states is approximately Gaussian as is illustrated by the fitted Gaussian in \figref{1}{B}.} \p{\figref{1}{A,B} illustrates that essentially all of the injected \beta B2-crystallin molecules have been measured. The measured amounts of proteins with different isotopic compositions and charge clearly approach zero on either side of the distribution functions, so there are no significant amounts of unmeasured types of proteins. This differs from the usual situation with peptide ionization, wherein the distribution functions usually include molecules with very low numbers of ions, thus allowing the possibility of unionized molecules.} \p{These experimentally measured curves were similar for all 14 crystallins. Therefore, this method of quantitative analysis is exceptionally reliable. A correctable sigmoidal variation in detector response with quantity of protein has been reported [11] for long scan times in 7 Tesla mass spectrometers. This variation was not observed in the isotopic abundances of the 12 Tesla measurements reported herein.} \p{The mass spectrometer measures the ratio of mass/charge for each ion. For instance, the eleventh isotope peak of the +22 ions of \beta B2-crystallin appears at a mass/charge of 1059.45=[23,276+(22)(1.008)+10]/22. The eleventh isotopic peak has mass increased by 10 Da through isotopic composition. The addition of 22 in the equation arises from the increase in mass by 1 Da from the addition of a proton with each positive charge.} \p{Multiplication of the masses of the observed 360 ions of \beta B2-crystallin and those of the other crystallins by their individual charges results in a spectrum of mass alone as is illustrated for a 2-day-old human lens sample in \figref{2}. The peaks shown appear to be unimodal, but they are actually isotopic envelopes as is shown for \alpha A-crystallin in the inset.} \p{The amounts of each protein in each lens sample were determined by summing the areas of all approximately 300 charge-corrected isotopic peaks for each protein. Within each lens sample, the initial percentage protein values were then concentration normalized by division by the sum of the amounts of \alpha A-, \alpha B-, \gamma D-, \gamma C-, and \beta B2-crystallin, which constitute about two-thirds of the total protein. This corrects for variations in the overall concentrations of the lens samples. All of the protein values were then multiplied by 66.67 to give value sums of approximately 100%. These five peaks were chosen for the normalization basis because of their large and relatively narrow distributions of amounts. These normalization operations were performed once on the entire data set before any further evaluation of the data was carried out. The values were not subsequently changed in any way.} \results \subsection{Normalized molar percentages of the crystallins} \p{\tabref{1} lists the median, mean, and percentage standard deviation of the mean of the normalized molar percentages of the 14 crystallins for three data sets: the four 2-day-old lenses; the set of 23, 25, 30, 150, and 300-day-old lenses; and the complete set of nine lenses. Listed separately are the values for the 90-day-old lens. This lens showed a sharply lower amount of \beta B2-crystallin and markedly higher amounts of \alpha B-, \gamma C-, and \gamma S-crystallin and is, therefore quite different from the other nine lens samples. This may be the result of unusual biological variation. The 90-day-old sample was, therefore, omitted from further data analysis. This donor died of sudden infant death syndrome and was on a ventilator prior to death.} \p{The percentages of the individual crystallins are remarkably narrowly distributed. Omitting crystallins with percentages under 4% where experimental error is expected to be higher, the average percentage standard deviation of the means of \alpha A-, \alpha B-, \gamma D-, \gamma C-, \gamma S-, \beta A4-, \beta B2-, and \beta B1-crystallin for the four 2-day-old lenses is 16%. For the 11, 23, 25, 30, 150, and 300-day-old lenses and for the entire set of nine lenses, these values are 22% and 22%, respectively.} \p{These percentages include the sum of all experimental errors of the measurement technique and all biological variation between the individual lenses. If experimental errors are estimated at least at 5%, average biological variation from individual to individual in the relative amounts of the crystallins at birth results in a standard deviation of the mean of no more than 10%, as calculated from the four two-day-old samples.} \p{\tabref{1} also lists the values from an earlier quantitative study [2] in which 0-, 3-, 4-, and 7-day-old human lenses were evaluated by replicated commasie blue staining and densitometric scanning of 2-D gels. These values have been normalized in the same manner as the current data. Considering the lesser accuracy of the gel, staining, and scanning method, our results are in acceptable agreement with the earlier study.} \subsection{Age-dependent trends for \alpha A- and \beta B2-crystallins} \p{\figref{3} and \figref{4} show significant percentage composition trends with age that are present for the \alpha A- and \beta B2-crystallins. \alpha A-crystallin decreases with age, and \beta B2-crystallin increases with age. The trends are approximately logarithmic with age. The squared correlation coefficients for the age logarithmic plots are 0.79 for \alpha A-crystallin and 0.76 for \beta B2-crystallin. In comparison, these coefficients for age linear plots are 0.49 for \alpha A- and 0.65 for \beta B2-crystallin, so the log plots are a better fit to the data than the linear plots. The percentage changes for the two proteins are offsetting, identical, and comparable to the increase of lens size expected between 2 and 300 days, which is also nonlinear [12]. These changes suggest that \beta B2-crystallin may be substituting for \alpha A-crystallin in lens fiber cells synthesized after birth.} \p{The decrease in \alpha A-crystallin between 2 days and 300 days is not primarily caused by postsynthetic modifications. Many postsynthetically modified crystallins were measured in these mass spectra, but these measurements are beyond the scope of this report. In the case of \alpha A-crystallin, the sum of all higher mass addition products such as oxidized, phosphorylated, and oxidized and phosphorylated \alpha A-crystallin increased by 2% relative to \alpha A-crystallin between 2 and 300 days, while the sum of lower mass deletion products such as truncated serine \alpha A-crystallin increased by 3% relative to \alpha A-crystallin. Simultaneously, between 2 days and 300 days, the molar percentage of \alpha A-crystallin decreased by 40%.} \subsection{Integer relationships between lens crystallins} \p{\figref{5} shows remarkable integral relationships between the median normalized molar percentages of the 14 crystallins in the 2-day-old lenses. \figref{5} summarizes these findings of integer relationships on a quantitative axis, so that the closeness of linear fit is graphically illustrated. These relationships were discovered after the data calculations were completed. No additional calculations or adjustments of calculations were thereafter performed. Also shown in \figref{5} are the \alpha A- and \beta B2-crystallin values for the 300-day-old lens.} \p{It is evident that there are six groups of crystallins that have quantitative integral relationships to each other in the ratios of 1/2, 1, 2, 3, 4, and 10 in the 2-day-old lens. The groups include \beta A2-, \beta A3-22-, and \beta B3-crystallin; \beta A1-, \beta A3-, and \beta B1-15-crystallin; \alpha B-, \gamma D-, and \beta A4-crystallin; \gamma S- and \beta B1-crystallin; \gamma C- and \beta B2-crystallin; and \alpha A-crystallin, respectively. Between 2 days and 300 days, \alpha A-crystallin diminishes from 10 to 6, while \beta B2-crystallin increases from 4 to 8, thus quantitatively offsetting \alpha A-crystallin. The means and standard deviations of the measured ratios in the 2-day-old crystallin groups are shown in \tabref{2}.} \p{Since \beta A3-22- and \beta B1-15-crystallins are post-translational modifications of \beta A3- and \beta B1-crystallin, \figref{5} could be constructed with these pairs combined. In that case, the \beta A3-crystallin combination would form a new group at integer 1.5, and the \beta B1-crystallin combination would join \gamma C- and \beta B2-crystallin at integer 4.} \p{It is interesting to note that the summed \alpha-, \beta-, and \gamma-crystallin protein forms have ratios to one another of 12:13.5:9. If \beta-crystallins at 1 or less after normalization and \alpha B-crystallin at 2 were involved in separate structures, this ratio for the remaining array would be 10:9:9. Furthermore, as summarized in \tabref{3}, the \beta- and \gamma-crystallins seem to be more closely similar to one another than with the \alpha-crystallins. The \alpha-crystallins include one 10 and one 2, while the \beta- and \gamma-crystallin each include one each of 2, 3, and 4 and the \beta-crystallin several smaller components. This is summarized in \tabref{3}. Therefore, there are approximately equal quantities of \alpha-, \beta-, and \gamma-crystallins in the newborn human lens.} \discussion \p{The relative in vivo normalized molar percentages of the 14 human lens crystallins measured herein are remarkable in three ways. First, in 2-day-old lenses, these molar percentages are very narrowly distributed for each crystallin. The mean percentage standard deviation for the 8 most abundant crystallins is 16%. This includes all experimental measurement errors and all biological individuality combined. This percentage rises to 22% when lenses of all ages between 2 and 300 days are pooled. Second, the percentages of \alpha A- and \beta B2-crystallin change during the first 300 days after birth by offsetting amounts that are comparable to the amount of lens growth expected during this time. We have previously observed a decrease in \alpha A-crystallin during aging [6]. These offsetting changes could arise in several ways. A simple way would be for \beta B2-crystallin to be synthesized instead of \alpha A-crystallin, thereby replacing it in lens crystallin arrays synthesized after birth.} \p{Third, the molar percentages of the 14 crystallins in the 2-day-old lens are found to have integral relationships to one another in the ratios of 1/2, 1, 2, 3, 4, and 10 that are far too pronounced to have arisen by chance. Further, the molar percentages of the \alpha-, \beta-, and \gamma-crystallin groups appear to have integral relationships with one another. These percentages probably reflect an ordered structure that exists at birth and is composed of these 14 different proteins. These ratios may be specific for the human lens. The rat lens, for example, has been reported to have quite different ratios [13]. Two of the 14 proteins are truncated \beta-crystallins that are detected even in the newborn and may reflect the age of the proteins in the older fiber cells found in the nucleus of the lens.} \p{\figref{5} shows the molar percentages of the 14 crystallins that were measured in these experiments in the eye lenses of 2-day-old human subjects. Values for the additional age ranges up to 300-day-old humans are listed in \tabref{1}. The molar percentages in the lenses of 2-day-old subjects for all 14 crystallins were found to lie very near to one of the seven values of 1.5, 3, 6, 9, 12, or 30. In the 300-day-old lens, the molar percentage of \beta B2-crystallin has shifted from 12 to 24, while \alpha A-crystallin has shifted from 30 to 18, exactly offsetting the \beta B2-crystallin shift of 12. Dividing each of the molar percentages by 3 (\figref{5} and \tabref{2}), shows that the molar ratios of the crystallins to one another are 0.5:1:2:3:4:10. \tabref{3} summarizes these by \alpha-, \beta-, and \gamma-crystallin types.} \p{So, the 14 crystallins of the newborn human eye lens are found to be present in molar ratios that bear the simple integer relationship of 0.5 to 1 to 2 to 3 to 4 to 10 with one another. These molecules are probably packed very closely with one another in the lens in ordered 3-dimensional arrays controlled by multiple specific mutual binding regions on each protein. The discovery of these integer relationships suggests both that these arrays are highly ordered and that their structures are such as to require molar amounts of each crystallin in specific ratios to the others.} \p{Fourier transform ion cyclotron resonance mass spectrometry was first applied to lens crystallin studies for the purpose of measuring crystallin deamidation [11]. In that application, direct injection of lens homogenates followed by quantitative measurement was proved feasible.} \p{This technique has now been applied herein to the quantitative measurement of the amounts of crystallins in human lenses as a function of age during the first year after birth. This technique has three principal advantages. First, it utilizes lens homogenates that are directly injected into the mass spectrometer with no prior sample preparation or fractionation. This avoids procedures that may compromise quantitation due to absorptive or other losses. Second, this technique entirely resolves the crystallins from one another, which avoids loss of quantitation from cross contamination. Third, virtually every injected crystallin molecule is ionized and quantitatively measured by ion image current in such a way that the resulting measurements are correct and based upon physical principles that are inherently linear and exact.} \p{Thus, this technique far surpasses, in convenience and accuracy, the various methods that depend upon wet chemistry separations of the crystallins. These advantages have permitted, in this initial application, the discovery of quantitative relationships between the crystallins that have been heretofore unknown.} \acknowledgements \p{We thank Dr. Christoph Borchers and the University of North Carolina for the use of the 12 Tesla FTMS instrument, National Institutes of Health grant EY 12239 to K. J. Lampi, and grants to the Oregon Institute of Science and Medicine by the Kinsman Foundation and the Morse Foundation for support of this work.} \references \p{1. Bloemendal H. The vertebrate eye lens. Science 1977; 197:127-38. \pubmed{877544}} \p{2. Lampi KJ, Ma Z, Shih M, Shearer TR, Smith JB, Smith DL, David LL. Sequence analysis of betaA3, betaB3, and betaA4 crystallins completes the identification of the major proteins in young human lens. J Biol Chem 1997; 272:2268-75. \pubmed{8999933}} \p{3. Lapko VN, Smith DL, Smith JB. Expression of betaA2-crystallin in human lenses. Exp Eye Res 2003; 77:383-5. \pubmed{12907171}} \p{4. Hanson SR, Hasan A, Smith DL, Smith JB. The major in vivo modifications of the human water-insoluble lens crystallins are disulfide bonds, deamidation, methionine oxidation and backbone cleavage. Exp Eye Res 2000; 71:195-207. \pubmed{10930324}} \p{5. Kim YH, Kapfer DM, Boekhorst J, Lubsen NH, Bachinger HP, Shearer TR, David LL, Feix JB, Lampi KJ. Deamidation, but not truncation, decreases the urea stability of a lens structural protein, betaB1-crystallin. Biochemistry 2002; 41:14076-84. \pubmed{12437365}} \p{6. Lampi KJ, Ma Z, Hanson SR, Azuma M, Shih M, Shearer TR, Smith DL, Smith JB, David LL. Age-related changes in human lens crystallins identified by two-dimensional electrophoresis and mass spectrometry. Exp Eye Res 1998; 67:31-43. \pubmed{9702176}} \p{7. Lund AL, Smith JB, Smith DL. Modifications of the water-insoluble human lens alpha-crystallins. Exp Eye Res 1996; 63:661-72. \pubmed{9068373}} \p{8. Zhang Z, Smith DL, Smith JB. Human beta-crystallins modified by backbone cleavage, deamidation and oxidation are prone to associate. Exp Eye Res 2003; 77:259-72. \pubmed{12907158}} \p{9. Takemoto LJ. Disulfide bond formation of cysteine-37 and cysteine-66 of beta B2 crystallin during cataractogenesis of the human lens. Exp Eye Res 1997; 64:609-14. \pubmed{9227279}} \p{10. Thomson JA, Augusteyn RC. Ontogeny of human lens crystallins. Exp Eye Res 1985; 40:393-410. \pubmed{4065234}} \p{11. Robinson NE, Lampi KJ, McIver RT, Williams RH, Muster WC, Kruppa G, Robinson AB. Quantitative measurement of deamidation in lens betaB2-crystallin and peptides by direct electrospray injection and fragmentation in a Fourier transform mass spectrometer. Mol Vis 2005; 11:1211-9 \mvref{11}{138}. \pubmed{16402021}} \p{12. Harding J. Cataract: biochemistry, epidemiology, and pharmacology. London: Chapman and Hall; 1991. p. 3. \lccn{90015007}} \p{13. Lampi KJ, Shih M, Ueda Y, Shearer TR, David LL. Lens proteomics: analysis of rat crystallin sequences and two-dimensional electrophoresis map. Invest Ophthalmol Vis Sci 2002; 43:216-24. \pubmed{11773034}} \endreferences \note_correction } \correction{ \p{22 June 2006:} \p{The paragraph before the subsection heading (in Results) "Integer relationships between lens crystallins" was styled as a heading and the heading itself was not. This has been corrected.} } \beginfigures \figfile{1}{ \figtitle{1} \p{Charge state and isotopic distributions for \beta B2-crystallin in one of the four 2-day-old human lenses. The charge state distribution (\panel{B}) shows that essentially all of the \beta B2-crystallin molecules received between 13 and 30 positive charges during passage through the nanospray mass spectrometer source. These quantities have been divided by the molecular charge in order to correct for the increased ion image current detection signal with increase in charge. Each of these charges, caused by the addition of single protons to the molecules, increases the mass of the protein by 1 Da. The absence from the distribution function shown in (\panel{B}) of protein molecules with 10 or fewer charges verifies that there are essentially no molecules with zero charge, so all have been ionized. The isotopic distribution of the 22+ charge \beta B2-crystallin ions (\panel{A}) shows that greater than 98% of the isotopic species are being measured and quantitatively summed, since only the smallest abundance isotopic peaks approaching zero relative abundance are lost in background noise on each side of the distribution. Taken together, (\panel{A}) and (\panel{B}) demonstrate that virtually all of the \beta B2-molecules are accounted for and measured by this ICR FTMS method.} \ctr{\gifimage{1}{500}{903}{71}} } \figfile{2}{ \figtitle{2} \p{Mass spectrum of a 2-day-old crystallin sample. This spectrum was constructed by multiplication of each protein species by its charge. Thus, the mass/charge output of the mass spectrometer is deconvoluted into a simple mass spectrum. Compression of the mass axis between 20,000 and 30,000 mass units gives the appearance of single modal peaks. Expansion of the scale, as illustrated for \alpha A-crystallin in the inset, shows the fine structure of the actual spectra. The superb resolution of the FTMS method when applied to unfractionated lens homogenates is demonstrated by this total mass spectrum. Integration of the peaks in this spectrum gives the values that are, after concentration normalization, listed in \tabref{1}. Small amounts of various post-synthetically modified crystallins are also observed in this spectrum. For example, oxidized, phosphorylated, and oxidized and phosphorylated \alpha A-crystallins are observed at masses 18, 80, and 98 Da higher than \alpha A-crystallin itself.} \ctr{\gifimage{2}{700}{1093}{76}} } \figfile{3}{ \figtitle{3} \p{Linear plot of the mole percentage of \alpha A-crystallin as a function of logarithm of age during the first year after birth. The percentage of \alpha A-crystallin falls from 30% at age 2 days to 18% at age 300 days. This is quantitatively compensated by an increase in \beta B2-crystallin as shown in \figref{4}. The open circle shows the value for the 90-day-old donor, which was excluded from the trend line and R\sup{2} calculation.} \ctr{\gifimage{3}{500}{864}{19}} } \figfile{4}{ \figtitle{4} \p{Linear plot of the mole percentage of \beta B2-crystallin as a function of logarithm of age during the first year after birth. The percentage of \beta B2-crystallin rises from 12% at age 2 days to 24% at age 300 days. This is quantitatively compensated by a decrease in \alpha A-crystallin as shown in \figref{3}. The open circle shows the value for the 90-day-old donor, which was excluded from the trend line and R\sup{2} calculation. This value is quite different from the others and may reflect unique biological variation.} \ctr{\gifimage{4}{500}{856}{18}} } \figfile{5}{ \figtitle{5} \p{Median molar percentages of the 14 crystallins in the lenses of four 2-day-old human donors and one 300-day-old donor. The percentage values are seen to be clustered in six groups including \beta A2-, \beta A3-22-, and \beta B3-crystallin; \beta A1-, \beta A3-, and \beta B1-15-crystallin; \alpha B-, \gamma D-, and \beta A4-crystallin; \gamma S- and \beta B1-crystallin; \gamma C- and \beta B2-crystallin; and \alpha A-crystallin. The means and standard deviations of these groups are listed in \tabref{2} and further summarized in \tabref{3}. They show even integral relationships to one another as illustrated by the 0 to 30 and 0 to 10 integer axes and red lines. Since \beta A3-22- and \beta B1-15-crystallin are truncated forms \beta A3- and \beta B1-crystallin, an alternative treatment would be to combine these respective pairs. When this is done, the integer relationships remain, while the respective positions in the integer array are changed as described herein. It is likely that the integral relationships between the crystallins shown in \figref{5} reflect the structures of ordered arrays of crystallins in lens fiber cells. If so, the translation messages required to synthesize the crystallins would also reflect these ratios and would correspond to characteristics of the individual crystallins and their intramolecular lens structures. The changes for \alpha A-crystallin and \beta B2-crystallin are equal, offsetting, and comparable to the lens growth expected between 2 and 300 days. They may reflect a substitution of \beta B2-crystallin for \alpha A-crystallin in lens arrays constructed after birth. The change shown for the 300-day-old lens is further verified by the trend of values for other older lenses as shown in \figref{3} and \figref{4}.} \ctr{\gifimage{5}{560}{745}{45}} } \begintables \tabfile{1}{ \tabtitle{1} \p{The Lampi et al. [2] previously reported percentages were normalized as described (The average of four donors, 0, 3, 4, and 7 days of age, were measured by scanning of 2-D gels). All crystallins in these samples and the 10 measured herein by FTMS were soluble. Only soluble proteins were analyzed because insoluble proteins were negligible due to the young ages of the donors. The 2 Day column shows a combination of four 2-day-old donors. Median and mean are mole percentages. Standard deviation is percent standard deviation of the mean value, so, mean=29.3 and percent standard deviation=11% represents mean=29.3\pom 3.2. The 23 day thru 300 day column shows the results of five 23, 25, 30, 150, and 300-day-old donors. The 2 day thru 300 day column shows the data from the combination of all nine donors. The 90 day column shows data indicating that the 90-day-old donor may represent an unusual biological variant. The asterisk indicates that \gamma S-crystallin co-migrated with \beta A1-crystallin for 15.4+% total. The double asterisks indicate that \beta A2-crystallin was not detected. The sharps (hash mark) indicate that \beta B3-crystallin co-migrated with \beta A3-crystallin for 6.2+% total.} \box{\pre{ 2 day 23 day-300 day 2 day-300 day 0, 3, 4, ----------------------- ----------------------- ----------------------- Crystallin Mass 7 day [2] Median Mean % St Dv Median Mean % St Dv Median Mean % St Dv 90 Day ---------- ----- --------- ------ ---- ------- ------ ---- ------- ------ ---- ------- ------ Alpha A 19939 25.5 29.5 29.3 11 19.5 20.8 14 24.3 24.6 22 22.7 Alpha B 20188 7.4 6.6 6.3 11 6.8 6.7 17 6.6 6.6 14 14.1 Gamma D 20594 2.9 5.8 5.5 29 7.7 7.8 9.2 7.1 6.7 24 8.6 Gamma C 20734 16.4 12.1 12.1 15 11.3 11.8 14 11.3 11.9 14 17.5 Gamma S 20904 Gamma S/ 8.6 8.6 8.6 8.9 8.8 5.4 8.9 8.7 6.7 13.1 Beta A1* Beta A2 21993 nd** 1.6 1.6 0.8 0.9 34 0.9 1.0 40 1.2 Beta A4 22271 5.0 6.7 6.5 17 5.2 5.4 59 6.3 5.9 41 8.6 Beta A3 22631 1.7 1.7 1.7 13 1.7 1.7 37 1.7 1.7 30 3.4 (23-215) Beta A1 23087 Gamma S/ 2.9 2.3 54 2.5 2.3 31 2.6 2.3 37 Beta A1* Beta B2 23276 14.6 12.7 13.4 21 19.5 19.6 19 17.3 16.8 27 3.8 Beta B3 24209 Beta A3/ 1.5 1.5 1.6 1.5 25 1.5 1.5 22 Beta B3# Beta A3 25176 Beta A3/ 3.0 3.4 51 4.2 3.8 30 3.2 3.6 38 Beta B3# Beta B1 26518 2.0 3.0 3.1 12 2.5 2.4 24 2.8 2.7 22 4.2 (16-251) Beta B1 27917 7.5 8.2 8.1 14 6.7 6.5 41 7.8 7.3 30 8.6 }} } \tabfile{2}{ \tabtitle{2} \p{Mean and standard deviation percentage values for the six groups of crystallins illustrated in \figref{5}. These means and variances show that these six groups are quantitatively distinct.} \ctr{\gifimage{2}{691}{98}{16}} } \tabfile{3}{ \tabtitle{3} \p{Numbers of crystallins having each integral percentage. The number of crystallins at each specific integer percentage is listed.} \ctr{\gifimage{3}{600}{79}{13}} }