Relative quantitative comparisons of the extracellular protein profiles of Staphylococcus aureus UAMS-1 and its sarA, agr, and sarA agr regulatory mutants using one-dimensional polyacrylamide gel electrophoresis and nanocapillary liquid chromatography coupled with tandem mass spectrometry
Relative quantitative comparisons of the extracellular protein profiles of Staphylococcus aureus UAMS-1 and its sarA, agr, and sarA agr regulatory mutants using one-dimensional polyacrylamide gel electrophoresis and nanocapillary liquid chromatography coupled with tandem mass spectrometry
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DOI：
10.1128/jb.00383-08
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发表时间：
2008-08-01
影响因子：
3.2
通讯作者：
Hart, Mark E.
中科院分区：
文献类型：
--
作者：
Jones, Richard C.;Deck, Joanna;Hart, Mark E.
一维聚丙烯酰胺凝胶电泳，然后用纳米毛细管液相色谱-质谱联用分析蛋白质分离金黄色葡萄球菌UAMS-1后3，6，12，和24 111的体外生长。使用称为归一化肽数的定量值确定蛋白质丰度，并且总体而言，已知与细胞壁相关的蛋白质在生长早期更丰富，而已知分泌到周围环境中的蛋白质在生长后期更丰富。此外，蛋白质从用过的培养基和细胞裂解物的菌株UAMS-1和其同基因sarA，agr，和sarA agr调节突变株在指数增长进行了鉴定，并比较其相对丰度。已知由全局调节剂sarA和agr调节的细胞外蛋白质显示与已知的关于这些调节剂的作用的蛋白质水平一致。例如，半胱氨酸蛋白酶（SspB）、内肽酶（SspA）、葡萄球菌蛋白酶（ScpA）和金霉素溶素（Aur）在sarA和sarA agr突变体中的丰度高于菌株UAMS-1。免疫球蛋白G（IgG）结合蛋白（Sbi），免疫显性葡萄球菌抗原A（IsaA），IgG结合蛋白A（Spa），血红素铁结合蛋白（IsdA）是最丰富的agr突变背景。在sarA突变体中其丰度降低的蛋白质包括纤维蛋白原结合蛋白（Fib [Efb]）、IsaA、脂肪酶I和2以及两种被鉴定为双组分白细胞毒素家族的推定杀白细胞素F和S亚基的蛋白质。总的来说，这种方法鉴定了1，263种蛋白质（两种或更多种肽的匹配），并提供了一种鉴定蛋白质和比较其相对丰度的方便可靠的方法。
One-dimensional polyacrylamide gel electrophoresis followed by nanocapillary liquid chromatography coupled with mass spectrometry was used to analyze proteins isolated from Staphylococcus aureus UAMS-1 after 3, 6, 12, and 24 111 of in vitro growth. Protein abundance was determined using a quantitative value termed normalized peptide number, and overall, proteins known to be associated with the cell wall were more abundant early on in growth, while proteins known to be secreted into the surrounding milieu were more abundant late in growth. In addition, proteins from spent media and cell lysates of strain UAMS-1 and its isogenic sarA, agr, and sarA agr regulatory mutant strains during exponential growth were identified, and their relative abundances were compared. Extracellular proteins known to be regulated by the global regulators sarA and agr displayed protein levels in accordance with what is known regarding the effects of these regulators. For example, cysteine protease (SspB), endopeptidase (SspA), staphopain (ScpA), and aureolysin (Aur) were higher in abundance in the sarA and sarA agr mutants than in strain UAMS-1. The immunoglobulin G (IgG)-binding protein (Sbi), immunodominant staphylococcal antigen A (IsaA), IgG-binding protein A (Spa), and the heme-iron-binding protein (IsdA) were most abundant in the agr mutant background. Proteins whose abundance was decreased in the sarA mutant included fibrinogen-binding protein (Fib [Efb]), IsaA, lipase I and 2, and two proteins identified as putative leukocidin F and S subunits of the two-component leukotoxin family. Collectively, this approach identified 1,263 proteins (matches of two peptides or more) and provided a convenient and reliable way of identifying proteins and comparing their relative abundances.