Tsiatsiani Liana, Giansanti Piero, Scheltema Richard A, van den Toorn Henk, Overall Christopher M, Altelaar A F Maarten, Heck Albert J R
Centre for Blood Research, Department of Oral Biological and Medical Sciences, and Department of Biochemistry and Molecular Biology, University of British Columbia , Vancouver V6T 1Z3, BC, Canada.
J Proteome Res. 2017 Feb 3;16(2):852-861. doi: 10.1021/acs.jproteome.6b00825. Epub 2016 Dec 2.
A key step in shotgun proteomics is the digestion of proteins into peptides amenable for mass spectrometry. Tryptic peptides can be readily sequenced and identified by collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD) because the fragmentation rules are well-understood. Here, we investigate LysargiNase, a perfect trypsin mirror protease, because it cleaves equally specific at arginine and lysine residues, albeit at the N-terminal end. LysargiNase peptides are therefore practically tryptic-like in length and sequence except that following ESI, the two protons are now both positioned at the N-terminus. Here, we compare side-by-side the chromatographic separation properties, gas-phase fragmentation characteristics, and (phospho)proteome sequence coverage of tryptic (i.e., (X)K/R) and LysargiNase (i.e., K/R(X)) peptides using primarily electron-transfer dissociation (ETD) and, for comparison, HCD. We find that tryptic and LysargiNase peptides fragment nearly as mirror images. For LysargiNase predominantly N-terminal peptide ions (c-ions (ETD) and b-ions (HCD)) are formed, whereas for trypsin, C-terminal fragment ions dominate (z-ions (ETD) and y-ions (HCD)) in a homologous mixture of complementary ions. Especially during ETD, LysargiNase peptides fragment into low-complexity but information-rich sequence ladders. Trypsin and LysargiNase chart distinct parts of the proteome, and therefore, the combined use of these enzymes will benefit a more in-depth and reliable analysis of (phospho)proteomes.
鸟枪法蛋白质组学中的一个关键步骤是将蛋白质消化成适合质谱分析的肽段。由于胰蛋白酶消化产生的肽段的裂解规则已被充分理解，因此可以通过碰撞诱导解离（CID）或高能碰撞解离（HCD）轻松地对其进行测序和鉴定。在此，我们研究了LysargiNase，一种完美的胰蛋白酶镜像蛋白酶，因为它在精氨酸和赖氨酸残基处具有同样特异性的切割作用，尽管是在N端。因此，LysargiNase肽段在长度和序列上实际上类似于胰蛋白酶肽段，只是在电喷雾电离（ESI）之后，两个质子现在都位于N端。在这里，我们主要使用电子转移解离（ETD）并与HCD进行比较，并排比较了胰蛋白酶（即(X)K/R）和LysargiNase（即K/R(X)）肽段的色谱分离特性、气相裂解特征以及（磷酸化）蛋白质组序列覆盖率。我们发现胰蛋白酶和LysargiNase肽段的裂解几乎呈镜像关系。对于LysargiNase，主要形成N端肽离子（ETD中的c离子和HCD中的b离子），而对于胰蛋白酶，在互补离子的同源混合物中，C端碎片离子占主导（ETD中的z离子和HCD中的y离子）。特别是在ETD过程中，LysargiNase肽段裂解成低复杂性但信息丰富的序列阶梯。胰蛋白酶和LysargiNase描绘了蛋白质组的不同部分，因此，联合使用这些酶将有助于对（磷酸化）蛋白质组进行更深入、可靠的分析。
