Che F Y, Eipper B A, Mains R E, Fricker L D
Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Cell Mol Biol (Noisy-le-grand). 2003 Jul;49(5):713-22.
We previously described a method of quantitating levels of peptides in Cpe(fat)/Cpe(fat) mice using affinity chromatography to isolate peptide-processing intermediates and differential isotopic labeling/mass spectrometry. In the present study, we compared two different isotopic labels, acetic anhydride and succinic anhydride for detection and quantitation of peptides in wild type mice. As previously found for acetic anhydride, succinic anhydride efficiently labels all primary amines in various peptides. Of these two reagents, succinic anhydride provides better resolution between the heavy and light peaks of the labelled peptides due to a greater mass difference between the deuterated (heavy) and non-deuterated (light) form of this label (4 Da for succinate, 3 Da for acetate). Using succinic anhydride labeling, the accuracy of measuring 1:1 and 1:2 ratios of peptides in pituitary extracts was within 5% of the theoretical value for most peptides. The accuracy with succinic anhydride is comparable to the accuracy of acetic anhydride and more peptides could be detected and quantitated with succinic anhydride. The two labels were then used to examine pituitary peptides in mice with a defect in copper transport (Atp7a mice) vs wild type mice. Using succinic anhydride, 13 peptides could be detected, 12 of which matched the theoretical mass of known pituitary peptides. Five of the six peptides which contain C-terminal amide groups were significantly decreased in the Atp7a mice relative to wild type mice, whereas only one non-amidated peptide was significantly decreased in Atp7a mice. With acetic anhydride, only five peptides could be quantitated. The three peptides which contain C-terminal amide groups were decreased approximately 30% in the Atp7a mice. The selective decrease in amidated peptides in Atp7a mice is consistent with the copper-requirement of the enzyme that forms C-terminal amides.
我们之前描述了一种使用亲和色谱法分离肽加工中间体并结合差异同位素标记/质谱法来定量Cpe(fat)/Cpe(fat)小鼠体内肽水平的方法。在本研究中，我们比较了两种不同的同位素标记物——乙酸酐和琥珀酸酐，用于检测和定量野生型小鼠体内的肽。正如之前对乙酸酐的研究发现，琥珀酸酐能有效标记各种肽中的所有伯胺。在这两种试剂中，琥珀酸酐在标记肽的重峰和轻峰之间提供了更好的分辨率，因为该标记物的氘代（重）和非氘代（轻）形式之间的质量差异更大（琥珀酸盐为4 Da，乙酸盐为3 Da）。使用琥珀酸酐标记，垂体提取物中肽的1:1和1:2比例测量的准确性对于大多数肽而言在理论值的5%以内。琥珀酸酐的准确性与乙酸酐相当，并且使用琥珀酸酐可以检测和定量更多的肽。然后，这两种标记物被用于检测铜转运缺陷小鼠（Atp7a小鼠）与野生型小鼠垂体中的肽。使用琥珀酸酐，可以检测到13种肽，其中12种与已知垂体肽的理论质量相符。相对于野生型小鼠，Atp7a小鼠中六个含有C末端酰胺基团的肽中有五个显著减少，而Atp7a小鼠中只有一个非酰胺化肽显著减少。使用乙酸酐时，只能定量五种肽。Atp7a小鼠中三个含有C末端酰胺基团的肽减少了约30%。Atp7a小鼠中酰胺化肽的选择性减少与形成C末端酰胺的酶对铜的需求一致。
