Proteome Center Rostock, University Medicine Rostock and University of Rostock, Schillingallee 69, 18059 Rostock, Germany.
TRANSCEND Centre, Regional Institute of Oncology (IRO) Iasi, Str. General Henri Mathias Berthelot Nr. 2-4, 700483 Iasi, Romania.
J Am Soc Mass Spectrom. 2023 Feb 1;34(2):171-181. doi: 10.1021/jasms.2c00244. Epub 2023 Jan 19.
With Intact Transition Epitope Mapping-Thermodynamic Weak-force Order (ITEM-TWO) analysis in combination with molecular modeling, the phosphorylation-dependent molecular recognition motif of the anti-HpTGEKP antibody has been investigated with binary and ternary component mixtures consisting of antibody and (phospho-) peptides. Amino acid sequences have been selected to match either the antibody's recognition motif or the cancer-related zinc finger protein mutations and phosphorylations of the respective amino acid residues. Upon electrospraying of all the components of the mixtures, that is, hexapeptides, antibody, and intact immune complexes, the produced ions were subjected to mass spectrometric mass filtering. The antibody ions as well as the immune complex ions traversed into the mass spectrometer's collision chamber, whereas paths of unbound peptide ions were blocked prior to entering the collision cell. After dissociation of the multiply charged immune complexes in the gas phase, the complex-released peptide ions were recorded after having traversed the second mass filter. Complex-released peptides were unambiguously identified by their masses using mass analysis with isotope resolution. From the results of our studies with seven (phospho-) peptides with distinct amino acid sequences, which resembled either the antibody's binding motif or mutations, we conclude the following: (i) A negatively charged phospho group, located near the peptide's N-terminus is mandatory for antibody binding when placed on the peptide surface at a precise distance to the C-terminally located positively charged ε-amino group of a lysinyl residue. (ii) A bulky amino acid residue, such as the tyrosinyl residue at the N-terminal position of the (phospho-) threoninyl residue, abolishes antibody binding. (iii) Two closely spaced phospho groups negatively interfere with the surface polarity pattern and abolish antibody binding as well. (iv) Non-phosphorylated peptides are not binding partners of the anti-HpTGEKP antibody.
采用完整过渡表位作图-热力学弱力有序（ITEM-TWO）分析联合分子建模，研究了抗-HpTGEKP 抗体的磷酸化依赖性分子识别基序，涉及由抗体和（磷酸化）肽组成的二元和三元混合物。选择氨基酸序列与抗体的识别基序或癌症相关锌指蛋白突变和相应氨基酸残基的磷酸化匹配。对混合物的所有成分（即六肽、抗体和完整免疫复合物）进行电喷雾后，产生的离子被进行质谱质量过滤。抗体离子以及免疫复合物离子进入质谱仪的碰撞室，而未结合的肽离子的路径在进入碰撞池之前被阻断。在气相中使多电荷免疫复合物解离后，在穿过第二个质量过滤器后记录释放出的复合物肽离子。使用具有同位素分辨率的质量分析，通过其质量明确识别复合物释放的肽。通过对七种具有不同氨基酸序列的（磷酸化）肽的研究，这些肽类似于抗体的结合基序或突变，我们得出以下结论：（i）当位于肽表面上且与赖氨酸的ε-氨基基团位于精确距离处时，位于肽 N 端附近的带负电荷的磷酸基团对于抗体结合是必需的。（ii）在 N 端位置的酪氨酸等大体积氨基酸残基会破坏抗体结合。（iii）两个紧密间隔的磷酸基团会强烈干扰表面极性模式并破坏抗体结合。（iv）非磷酸化肽不是抗-HpTGEKP 抗体的结合伴侣。
