Department of Chemistry, The University of Adelaide, South Australia, 5005, Australia.
Rapid Commun Mass Spectrom. 2013 Nov 15;27(21):2287-96. doi: 10.1002/rcm.6686.
To determine the negative-ion cleavages from M-H ions of Ser sulfate-containing peptides using experiment and theory in concert.
Fragmentations were explored using a Waters QTOF2 mass spectrometer in negative-ion electrospray mode, together with calculations at the CAM-B3LYP/6-311++g(d,p) level of theory. Peptides used in this study were: GS(SO3H)(OH) 1 GS(SO3H)(OCH3) 1a GAVS(SO3H)(OH) 2 GAVS(SO3H)(OCH3) 2a GLS(SO3H)(GVA(OH) 3 GLS(SO3H)GDA(OH) 4 GLS(SO3H)GS(SO3H)A(OH) 5.
Previously, it has been shown that a peptide containing a Tyr sulfate group shows [(M-H)(-) -SO3] as the base peak. Only a small peak was observed corresponding to HOSO3(-) (formed following rearrangement of the sulfate). A Ser sulfate-containing peptide, in contrast, shows pronounced peaks due to cleavage product anions [(M-H)(-)-SO3] and HOSO3(-). Theoretical calculations at the CAM-B3LYP/6-311++g(d,p) level of theory suggest that rearrangement of a Ser sulfate to give C-terminal CO2SO3H is energetically unfavourable in comparison with fragmentation of the intact Ser sulfate to yield [(M-H)(-)-SO3] and HOSO3(-). [(M-H)(-)-H2SO4] anions are not observed in the spectra of peptides containing Ser sulfate, presumably because HOSO3(-) is a relatively weak gas-phase base (ΔGacid = 1265 kJ mol(-1)).
Experimental and theoretical data suggest that [(M-H)(-)-SO3] and HOSO3(-) product anions (from a peptide with a C-terminal Ser sulfate) are formed from the serine sulfate anion accompanied by specific proton transfer. CID MS/MS/MS data for an [(M-H)(-)-SO3] ion of an underivatised sulfate-containing peptide will normally allow the determination of the amino acid sequence of that peptide. The one case we have studied where that is not the case is GLS(SO3H)GDA(OH), where the peptide contains Ser sulfate and Asp, where the diagnostic Asp cleavages are competitive with the Ser sulfate cleavages.
通过实验和理论相结合，确定含有丝氨酸硫酸盐的肽[M-H]（-）离子的负离子裂解。
使用 Waters QTOF2 质谱仪在负离子电喷雾模式下进行碎裂研究，并在 CAM-B3LYP/6-311++g(d,p)理论水平进行计算。本研究中使用的肽为：GS(SO3H)(OH)1GS(SO3H)(OCH3)1aGAVS(SO3H)(OH)2GAVS(SO3H)(OCH3)2aGLS(SO3H)(GVA(OH)3GLS(SO3H)GDA(OH)4GLS(SO3H)GS(SO3H)A(OH)5。
此前已表明，含有酪氨酸硫酸盐的肽显示[(M-H)(-) -SO3]作为基峰。仅观察到对应于 HOSO3(-)（硫酸盐重排后形成）的小峰。相比之下，含有丝氨酸硫酸盐的肽显示出明显的裂解产物阴离子[(M-H)(-)-SO3]和 HOSO3(-)的峰。CAM-B3LYP/6-311++g(d,p)理论水平的理论计算表明，与完整丝氨酸硫酸盐断裂生成[(M-H)(-)-SO3]和 HOSO3(-)相比，丝氨酸硫酸盐重排生成 C 末端 CO2SO3H 在能量上是不利的。[(M-H)(-)-H2SO4]阴离子在含有丝氨酸硫酸盐的肽的光谱中未观察到，可能是因为 HOSO3(-)是相对较弱的气相碱（ΔGacid = 1265 kJ mol(-1)）。
实验和理论数据表明，[(M-H)(-)-SO3]和 HOSO3(-)产物阴离子（来自末端丝氨酸硫酸盐的肽）是由丝氨酸硫酸盐阴离子伴随特定质子转移形成的。未衍生的含硫酸盐肽的[(M-H)(-)-SO3]离子的 CID MS/MS/MS 数据通常可以确定该肽的氨基酸序列。我们研究的一个例外是 GLS(SO3H)GDA(OH)，其中肽含有丝氨酸硫酸盐和天冬氨酸，其中诊断性天冬氨酸裂解与丝氨酸硫酸盐裂解竞争。
