Quantitative measurement of deamidation in lens betaB2-crystallin and peptides by direct electrospray injection and fragmentation in a Fourier transform mass spectrometer.
Quantitative measurement of deamidation in lens betaB2-crystallin and peptides by direct electrospray injection and fragmentation in a Fourier transform mass spectrometer.
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发表时间：
2005-12
期刊：
影响因子：
2.2
通讯作者：
N. Robinson;K. Lampi;R. T. McIver;R. Williams;W. C. Muster;G. Kruppa;A. B. Robinson
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文献类型：
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作者：
N. Robinson;K. Lampi;R. T. McIver;R. Williams;W. C. Muster;G. Kruppa;A. B. Robinson
透镜晶体蛋白的脱酰胺和特定的脱酰胺位点被认为与衰老和白内障有关。然而，这些研究受到缺乏合适的定量方法测量蛋白质脱酰胺的阻碍。我们在此证明了一种方法，用于定量测量蛋白质和肽的脱酰胺化，而无需预先制备或分离样品，以便直接比较酰胺化和脱酰胺化形式。我们已经测试了这一假设，即19 mDa的质量缺陷，区分脱酰胺肽和蛋白质从普通的天然同位素物种可以用于定量测量其脱酰胺的速率和程度。所使用的测量技术是离子回旋共振傅里叶变换质谱法（FTMS），单独没有事先的样品制备或分离。酰胺化和脱酰胺物质是重组表达的人眼透镜β B2-晶状体蛋白和肽GlyIleAsnAlaGly和GlyAsnAsnAsnGly。还对来自1个月大的人类供体的透镜蛋白进行了FTMS测量。方法在细菌中制备人眼透镜β B2-晶体蛋白（Gln 162被Glu 162取代），并采用梅里菲尔德固相多肽合成法合成GlyIleAsnAlaGly和GlyAsnAsnGly。在分析之前，将肽在pH 7.4、37.00 ℃、0.15 M Tris-HCl水溶液中脱酰胺18个连续的时间间隔。将不同组成百分比的突变体和野生型β B2-晶状体蛋白溶液混合并分析。通过电喷雾电离引入肽，并立即在离子回旋共振（ICR）傅立叶变换质量分析仪中进行分析。两个质量缺陷分析程序证明了蛋白质。首先，β B2-晶状体蛋白通过电喷雾电离引入质谱仪，+29同位素基团选择性地引入ICR质量分析仪，其中分离14个残基和18个残基激光诱导片段，并通过质量缺陷分析确定脱酰胺的程度。在第二种方法中，通过电喷雾电离将β B2-晶状体蛋白引入质谱仪，并在引入ICR质量分析仪之前通过碰撞电离将整个样品片段化，其中分离出14个残留片段，并通过质量缺陷分析确定脱酰胺程度。结果β B2-晶状体蛋白的质谱对脱酰胺程度有很好的定量依赖性。通过电喷雾电离直接进样，然后进行离子选择和激光裂解或碰撞裂解，产生适于FTMS定量分析的酰胺化和脱酰胺化β B2-晶体蛋白片段。两种肽显示出预期的四条脱酰胺速率曲线，精密度可接受。结论：本文首次报道了蛋白质脱酰胺的质量缺陷FTMS定量分析，并以β B2-晶状体蛋白为例进行了说明，这将证明是非常有用的。该方法省略了凝胶分离、色谱法、酶消化、衍生化和目前增加成本和时间同时降低酰胺化和脱酰胺化形式的定量比较的其它方法。质量缺陷FTMS也非常适合于肽的定量脱酰胺速率研究。该技术的实质性潜在意义是显而易见的，例如，对于透镜晶体蛋白，它使得迄今为止非常困难的年龄和疾病依赖性脱酰胺的定量研究成为可能。这种技术应该允许方便和可靠的识别和定量测量特定的脱酰胺位点，可能在老化和白内障中发挥作用。
PURPOSE Deamidation of lens crystallins and specific deamidation sites have been suggested to be associated with aging and cataracts. However, these studies have been hindered by the lack of suitable quantitative methods of measurement of protein deamidation. We demonstrate herein a method to quantitatively measure deamidation of proteins and peptides without prior sample preparation or separation in order to directly compare the amidated and deamidated forms. We have tested the hypothesis that the 19 mDa mass defect that distinguishes deamidated peptides and proteins from the ordinary natural isotopic species can be utilized for quantitative measurement of their rate and extent of deamidation. The measurement technique used was ion cyclotron resonance Fourier transform mass spectrometry (FTMS), alone with no prior sample preparation or separation. The amidated and deamidated species were recombinantly expressed human eye lens betaB2-crystallins and the peptides GlyIleAsnAlaGly and GlyAsnAsnAsnGly. FTMS measurements of lens proteins from a 1-month-old human donor were also carried out. METHODS Wild type and mutant human eye lens betaB2-crystallins with Gln162 replaced by Glu162 were produced in bacteria, and GlyIleAsnAlaGly and GlyAsnAsnAsnGly were synthesized by Merrifield solid-phase peptide synthesis. The peptides were deamidated in pH 7.4, 37.00 degrees C, 0.15 M Tris-HCl aqueous solution for 18 successive time intervals before analysis. Mutant and wildtype betaB2-crystallin solutions at various compositional percentages were mixed and analyzed. The peptides were introduced by electrospray ionization and immediately analyzed in the ion cyclotron resonance (ICR) Fourier transform mass analyzer. Two mass defect analysis procedures were demonstrated for the proteins. In the first, betaB2-crystallin was introduced into the mass spectrometer by electrospray ionization and the +29 isotopic group was selectively introduced into the ICR mass analyzer, where 14 residue and 18 residue laser-induced fragments were separated and the extent of deamidation determined by mass defect analysis. In the second, betaB2-crystallin was introduced into the mass spectrometer by electrospray ionization and the entire sample was fragmented by collision ionization before introduction into the ICR mass analyzer, where 14 residue fragments were separated and the extent of deamidation determined by mass defect analysis. RESULTS The betaB2-crystallin mass spectra showed a good quantitative dependence upon extent of deamidation. Direct injection by electrospray ionization followed by ion selection and laser fragmentation or by collision fragmentation produced fragments of amidated and deamidated betaB2-crystallin that were appropriate for FTMS quantitative analysis. The two peptides exhibited the expected four deamidation rate curves with acceptable precision. CONCLUSIONS Mass defect FTMS quantitative analysis of protein deamidation, as reported for the first time herein and illustrated with betaB2-crystallin, should prove quite useful. This procedure omits gel separation, chromatography, enzymatic digestion, derivatization, and other procedures that currently add cost and time while degrading quantitative comparison of the amidated and deamidated forms. Mass defect FTMS is also well suited to quantitative deamidation rate studies of peptides. The substantial potential significance of this technique is evident, as example, for lens crystallins where it makes possible quantitative studies of age and disease-dependent deamidation that have heretofore been very difficult. This technique should allow convenient and reliable identification and quantitative measurement of specific deamidation sites that may play a role in aging and cataracts.