Department of Genome Sciences, University of Washington, Seattle, WA 98195-5065, USA.
Anal Chem. 2010 Mar 15;82(6):2561-7. doi: 10.1021/ac1001433.
We describe a method to measure protein synthesis and catabolism in humans without prior purification and use the method to measure the turnover of surfactant protein-B (SP-B). SP-B, a lung-specific, hydrophobic protein essential for fetal-neonatal respiratory transition, is present in only picomolar quantities in tracheal aspirate samples and difficult to isolate for dynamic turnover studies using traditional in vivo tracer techniques. Using infusion of [5,5,5-(2)H(3)] leucine and a targeted proteomics method, we measured both the quantity and kinetics of SP-B tryptic peptides in tracheal aspirate samples of symptomatic newborn infants. The fractional synthetic rate (FSR) of SP-B measured using the most abundant proteolytic fragment, a 10 amino acid peptide from the carboxy-terminus of proSP-B (SPTGEWLPR), from the circulating leucine pool was 0.035 +/- 0.005 h(-1), and the fractional catabolic rate was 0.044 +/- 0.003 h(-1). This technique permits high-throughput and sensitive measurement of turnover of low abundance proteins with minimal sample preparation.
我们描述了一种无需事先纯化即可测量人类蛋白质合成和分解代谢的方法，并使用该方法测量表面活性蛋白-B (SP-B) 的周转率。SP-B 是一种肺特异性、疏水性蛋白，对胎儿-新生儿呼吸过渡至关重要，在气管抽吸样本中仅以皮摩尔数量存在，并且难以使用传统的体内示踪技术进行动态周转研究来分离。使用 [5,5,5-(2)H(3)] 亮氨酸输注和靶向蛋白质组学方法，我们测量了症状性新生儿气管抽吸样本中 SP-B 胰蛋白酶肽的数量和动力学。使用最丰富的蛋白水解片段（来自 proSP-B 的羧基末端的 10 个氨基酸肽 SPTGEWLPR）从循环亮氨酸池中测量的 SP-B 的分数合成率 (FSR) 为 0.035 +/- 0.005 h(-1)，而分数分解率为 0.044 +/- 0.003 h(-1)。该技术允许在最小的样品制备下，对低丰度蛋白质进行高通量和敏感的周转测量。
