Pereira Navaza Ana, Ruiz Encinar Jorge, Ballesteros Alfredo, González José M, Sanz-Medel Alfredo
Department of Physical and Analytical Chemistry, University of Oviedo, 33006 Oviedo, Spain.
Anal Chem. 2009 Jul 1;81(13):5390-9. doi: 10.1021/ac9005606.
The validity of using tyrosine iodination chemistry for the absolute and generic quantification of peptides by capillary high-performance liquid chromatography (capHPLC) coupled to inductively coupled plasma mass spectrometry (ICPMS) is investigated in detail. In this approach, two iodine atoms are specifically bioconjugated to the meta positions of the aromatic ring of every tyrosine residue. Characterization studies by capHPLC with parallel ICPMS and electrospray ionization tandem mass spectrometry (ESIMS/MS) detection clearly showed that such labeling iodination reaction affords one to obtain most accurate peptide determinations (after translation of the picomoles of iodine, quantified by ICPMS in each chromatographic peak, into picomoles of the corresponding labeled peptide). It is demonstrated that only, but every, tyrosine residue present in the peptide is completely diiodinated. The excellent detection limits for iodine using ICPMS allowed robust and highly sensitive tyrosine-containing peptide quantification (480 pM, 480 amol absolute). Derivatization is easily accomplished in a water/acetonitrile solution in only 2 min. Moreover, since the signal in ICPMS is completely independent from the chemical species containing the detected element, any iodine-containing standard (e.g., iodobenzoic acid) could be used as internal standard for the absolute quantification of every iodine-labeled tyrosine-containing peptide separated and detected along the gradient. The approach was optimized for tyrosine labeling and then validated by application to the absolute quantification of the three standard peptides present in the only reference material for peptide quantity (NIST 8327) commercially available. Identification of the species quantified by ICPMS was carried out by parallel capHPLC-ESI quadrupole time-of-flight (Q/TOF) analysis and corresponded, as expected, to the diiodinated peptides. The collision-induced dissociation (CID) spectra obtained demonstrated unequivocally the specific and complete derivatization of the tyrosine residues. The obtained quantitative results closely matched the reference values reported by the National Institute of Standards and Technology (NIST). In terms of precision, the relative standard deviation was as low as 3% RSD. Finally the approach was tested for the absolute quantification of proteins using a model standard protein (beta-casein). Results agreed again with the value specified showing that this labeling reaction is compatible with tryptic digestion.
详细研究了采用酪氨酸碘化化学方法，通过毛细管高效液相色谱（capHPLC）与电感耦合等离子体质谱（ICPMS）联用对肽进行绝对定量和通用定量的有效性。在这种方法中，两个碘原子特异性地生物共轭到每个酪氨酸残基芳香环的间位。通过capHPLC结合平行ICPMS和电喷雾电离串联质谱（ESIMS/MS）检测进行的表征研究清楚地表明，这种标记碘化反应能够最准确地测定肽（在将每个色谱峰中通过ICPMS定量的皮摩尔碘转化为相应标记肽的皮摩尔后）。结果表明，肽中存在的每个酪氨酸残基都能被完全二碘化。使用ICPMS对碘具有出色的检测限，从而能够对含酪氨酸的肽进行可靠且高度灵敏的定量（480 pM，绝对量480 amol）。衍生化在水/乙腈溶液中仅需2分钟即可轻松完成。此外，由于ICPMS中的信号完全独立于含有被检测元素的化学物种，任何含碘标准物（如碘苯甲酸）都可作为内标，用于对沿梯度分离和检测的每个碘标记的含酪氨酸肽进行绝对定量。该方法针对酪氨酸标记进行了优化，然后通过应用于商业上唯一可获得的肽定量参考物质（NIST 8327）中存在的三种标准肽的绝对定量进行了验证。通过平行capHPLC - ESI四极杆飞行时间（Q/TOF）分析对ICPMS定量分析的物种进行鉴定，结果如预期的那样，与二碘化肽相对应。获得的碰撞诱导解离（CID）光谱明确证明了酪氨酸残基的特异性和完全衍生化。获得的定量结果与美国国家标准与技术研究院（NIST）报告的参考值非常匹配。在精密度方面，相对标准偏差低至3% RSD。最后，使用模型标准蛋白（β - 酪蛋白）对该方法进行了蛋白质绝对定量测试。结果再次与指定值相符，表明这种标记反应与胰蛋白酶消化兼容。
