Sun Y, Bauer M D, Lu W
Corporate Research Division, Procter & Gamble Co., Miami Valley Laboratories, Cincinnati, Ohio 45253-8707, USA.
J Mass Spectrom. 1998 Oct;33(10):1009-16. doi: 10.1002/(SICI)1096-9888(1998100)33:10<1009::AID-JMS717>3.0.CO;2-4.
Penicillin-binding protein 2a (PBP2a), a high molecular mass PBP, is the primary enzyme responsible for the beta-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA). Inhibition of a PBP such as PBP2a by beta-lactams is due to covalent modification of an active site serine residue. Based on the sequence alignment with well studied beta-lactamases, DD-carboxypeptidases and other high molecular mass PBPs, the serine of a tetrad S403XXK in PBP2a was tentatively identified as the penicillin-binding site. However, direct evidence for the involvement of serine403 has not been reported. In this study, a method which combines liquid chromatography/electrospray mass spectrometry (LC/MS) and nano-electrospray MS for the identification of the active site serine in PBP2a is described. The covalent binding of the beta-lactams was carried out in vitro with the recombinant PBP2a. Peptide mapping of the cyanogen bromide fragments from penicilloyl-PBP2a, using microbore LC/MS, provided a rapid identification of the modified peptide with a 334 Da mass increase. The acylated peptide was isolated and further digested with trypsin. Nano-electrospray MS/MS sequencing of the acylated peptide in the tryptic digest showed that the penicillin was indeed attached to serine403.
青霉素结合蛋白2a（PBP2a）是一种高分子量的PBP，是耐甲氧西林金黄色葡萄球菌（MRSA）中β-内酰胺耐药性的主要负责酶。β-内酰胺对诸如PBP2a等PBP的抑制作用是由于活性位点丝氨酸残基的共价修饰。基于与已充分研究的β-内酰胺酶、DD-羧肽酶和其他高分子量PBPs的序列比对，PBP2a中四联体S403XXK的丝氨酸被初步鉴定为青霉素结合位点。然而，丝氨酸403参与其中的直接证据尚未见报道。在本研究中，描述了一种结合液相色谱/电喷雾质谱（LC/MS）和纳米电喷雾质谱来鉴定PBP2a中活性位点丝氨酸的方法。β-内酰胺的共价结合在体外与重组PBP2a进行。使用微径LC/MS对青霉酰-PBP2a的溴化氰片段进行肽图谱分析，能够快速鉴定出质量增加334 Da的修饰肽。分离出酰化肽并用胰蛋白酶进一步消化。胰蛋白酶消化物中酰化肽的纳米电喷雾MS/MS测序表明青霉素确实连接在丝氨酸403上。
