The α-granule proteome: novel proteins in normal and ghost granules in gray platelet syndrome
The α-granule proteome: novel proteins in normal and ghost granules in gray platelet syndrome
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DOI：
10.1111/j.1538-7836.2010.03932.x
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发表时间：
2010-08-01
影响因子：
10.4
通讯作者：
Gunay-Aygun, M.
中科院分区：
文献类型：
--
作者：
Maynard, D. M.;Heijnen, H. F. G.;Gunay-Aygun, M.
背景：血小板中颗粒结合物质的缺陷会导致先天性出血性疾病，称为储血池缺陷。对于灰血小板综合征（GPS）等疾病，其中观察到血小板减少症、血小板增大和α颗粒缺乏，仅定义了临床和组织学状态。目的：为了了解GPS中的分子缺陷，通过质谱法（MS）将来自正常个体的α-颗粒级分蛋白质组成与GPS患者的α-颗粒级分蛋白质组成进行比较。方法：采用蔗糖梯度离心法分离血小板细胞器。沉淀组分中的蛋白质通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳分离，还原，烷基化，并用胰蛋白酶消化。通过液相色谱-串联MS分析肽。Mascot用于肽/蛋白质鉴定并确定肽假阳性率。MassSieve用于生成和比较蛋白质的简约列表。结果如下：与对照组相比，归一化肽命中（NPH）从可溶性，生物合成的α-颗粒蛋白显着减少或未检测到GPS血小板，而NPH从可溶性，内吞α-颗粒蛋白只受到中度影响。正常血小板和GPS血小板中膜结合α颗粒蛋白的NPH相似，但P-选择素和Glut 3略有下降，与静息血小板中的免疫电镜结果一致。我们还确定了以前不知道在GPS中减少的蛋白质，包括潜在的转化生长因子-β结合蛋白1（LTBP 1），转化生长因子-β（TGF-β）复合物的一个组成部分。结论：我们的研究结果支持GPS中存在的“鬼颗粒”，点GPS的基本缺陷，未能将内源性合成的巨核细胞蛋白纳入α-颗粒，并确定特定的新蛋白质作为α-颗粒居民。
Background: Deficiencies in granule-bound substances in platelets cause congenital bleeding disorders known as storage pool deficiencies. For disorders such as gray platelet syndrome (GPS), in which thrombocytopenia, enlarged platelets and a paucity of alpha-granules are observed, only the clinical and histologic states have been defined. Objectives: In order to understand the molecular defect in GPS, the alpha-granule fraction protein composition from a normal individual was compared with that of a GPS patient by mass spectrometry (MS). Methods: Platelet organelles were separated by sucrose gradient ultracentrifugation. Proteins from sedimented fractions were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis, reduced, alkylated, and digested with trypsin. Peptides were analyzed by liquid chromatography-tandem MS. Mascot was used for peptide/protein identification and to determine peptide false-positive rates. MassSieve was used to generate and compare parsimonious lists of proteins. Results: As compared with control, the normalized peptide hits (NPHs) from soluble, biosynthetic alpha-granule proteins were markedly decreased or undetected in GPS platelets, whereas the NPHs from soluble, endocytosed alpha-granule proteins were only moderately affected. The NPHs from membrane-bound alpha-granule proteins were similar in normal platelets and GPS platelets, although P-selectin and Glut3 were slightly decreased, consistent with immunoelectron microscopy findings in resting platelets. We also identified proteins not previously known to be decreased in GPS, including latent transforming growth factor-beta-binding protein 1(LTBP1), a component of the transforming growth factor-beta (TGF-beta) complex. Conclusions: Our results support the existence of 'ghost granules' in GPS, point to the basic defect in GPS as failure to incorporate endogenously synthesized megakaryocytic proteins into alpha-granules, and identify specific new proteins as alpha-granule inhabitants.