Samgina Tatyana Yu, Vorontsov Egor A, Gorshkov Vladimir A, Artemenko Konstantin A, Zubarev Roman A, Lebedev Albert T
Department of Chemistry, Moscow State University, Russian Federation, 119991, Leninskie Gory 1/3, Moscow, Russia.
Rapid Commun Mass Spectrom. 2014 Dec 15;28(23):2595-604. doi: 10.1002/rcm.7049.
Mass spectrometry has shown itself as the most efficient tool for the sequencing of peptides. However, de novo sequencing of novel natural peptides is significantly more challenging in comparison with the same procedure applied for the tryptic peptides. To reach the goal in this case it is essential to select the most useful methods of triggering fragmentation and combine complementary techniques.
Comparison of low-energy collision-induced dissociation (CID) and higher energy collision-induced dissociation (HCD) modes for sequencing of the natural non-tryptic peptides with disulfide bonds and/or several proline residues in the backbone was achieved using an LTQ FT Ultra Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a 7 T magnet and an LTQ Orbitrap Velos ETD (Thermo Fisher Scientific, Bremen, Germany) instrument. Peptide fractions were obtained by high-performance liquid chromatography (HPLC) separation of frog skin secretion samples from ten species of Rana temporaria, caught in the Kolomna district of Moscow region (Russia).
HCD makes the b/y series longer and more pronounced, thus increasing sequence coverage. Fragment ions due to cleavages at the C-termini of proline residues make the sequencing more reliable and may be used to detect missed cleavages in the case of tryptic peptides. Another HCD peculiarity involves formation of pronounced inner fragment ions (secondary y(n)b(m) ion series formed from the abundant primary y-ions). Differences in de novo sequencing of natural non-tryptic peptides with CID and HCD, involving thorough manual expert interpretation of spectra and two automatic sequencing algorithms, are discussed.
Although HCD provides better results, a combination of CID and HCD data may notably increase reliability of de novo sequencing. Several pairs of b2 /a2 -ions may be formed in HCD, complicating the spectra. Automatic de novo sequencing with the available programs remains less efficient than the manual one, independently of the collision energy.
质谱已证明自身是肽测序最有效的工具。然而，与对胰蛋白酶肽段进行的相同操作相比，新型天然肽的从头测序具有显著更高的挑战性。在这种情况下，要实现目标，选择最有用的引发碎片化方法并结合互补技术至关重要。
使用配备7 T磁体的LTQ FT Ultra傅里叶变换离子回旋共振（FTICR）质谱仪（德国不来梅的赛默飞世尔科技公司）和LTQ Orbitrap Velos ETD仪器（德国不来梅的赛默飞世尔科技公司），比较低能量碰撞诱导解离（CID）和高能量碰撞诱导解离（HCD）模式对具有二硫键和/或主链中多个脯氨酸残基的天然非胰蛋白酶肽进行测序的效果。通过高效液相色谱（HPLC）分离从俄罗斯莫斯科地区科洛姆纳区捕获的10种欧洲林蛙的蛙皮分泌样品获得肽段组分。
HCD使b/y系列更长且更明显，从而增加序列覆盖率。脯氨酸残基C末端裂解产生的碎片离子使测序更可靠，并且在胰蛋白酶肽段的情况下可用于检测漏切。HCD的另一个特点涉及形成明显的内部碎片离子（由丰富的初级y离子形成的次级y(n)b(m)离子系列）。讨论了使用CID和HCD对天然非胰蛋白酶肽进行从头测序的差异，包括对光谱进行全面的人工专家解读以及两种自动测序算法。
尽管HCD提供了更好的结果，但CID和HCD数据的结合可显著提高从头测序的可靠性。HCD中可能形成几对b2 /a2离子，使光谱复杂化。无论碰撞能量如何，使用现有程序进行自动从头测序仍然不如人工测序高效。
