Method development for the concentration determination of a protein in human plasma utilizing 96-well solid-phase extraction and liquid chromatography/tandem mass spectrometric detection

Although liquid chromatography/tandem mass spectrometry (LC/MS/MS) technology has been widely used for quantitative analysis of small organic molecules, it has been a challenging task to quantitatively analyze protein samples utilizing this technology in biological matrices for pre-clinical and clinical studies. Here we present our initial results in method development for the quantitative determination of rK5 protein concentrations in human plasma samples utilizing LC/MS/MS technology. A protein similar in structure to rK5, but with a slightly reduced molecular weight, was used as internal standard. A 96-well solid-phase extraction procedure was developed to effectively extract protein analytes from plasma samples. Quantitative analysis was obtained by a novel approach of protein monitoring that employed selective reaction monitoring (SRM). Even though mass spectrometry of the internal standard protein gave no fragment ions, SRM monitoring greatly reduced background interference. Using samples prepared in human plasma with sodium EDTA as anticoagulant, a correlation coefficient (r(2)) of 0.9940 was obtained by producing a single standard curve with the injection of six rows of standards with a concentration range from 100 ng/mL to 10 microg/mL. The mean analytical recovery for these standards ranged from 91.5 to 103.6%. The CVs for individual standard levels ranged from 3.7 to 20.9%. The experiment was also repeated using samples prepared in human plasma with sodium heparin as anticoagulant, which produced a correlation coefficient (r(2)) of 0.9952 obtained from a single standard curve with the injection of four rows of standards with a concentration range from 50 ng/mL to 10 microg/mL. The mean analytical recovery for the standards ranged from 96.2 to 104.6%. The CVs for individual standard levels ranged from 2.6 to 15.6%.     

Two-dimensional gel electrophoresis maps of the proteome and phosphoproteome of primitively cultured rat mesangial cells

Mesangial cells (MC) play an important role in maintaining the structure and function of the glomerulus. The proliferation of MC is a prominent feature of many kinds of glomerular disease. The first reference 2-DE maps of rat mesangial cells (RMC), stained with silver staining or Pro-Q Diamond dye, have been established here to describe the proteome and phosphoproteome of RMC, respectively. A total of 157 selected protein spots, corresponding to 118 unique proteins, have been identified by MALDI-TOF-MS or LC-ESI-IT-MS/MS, in which 37 protein spots representing 28 unique proteins have also been stained with Pro-Q Diamond, indicating that they are in phosphorylated forms. All the identified proteins were bioinformatically annotated in detail according to their physiochemical characteristics, subcellular location, and function. Most of the separated or identified protein spots are distributed in the area of mass 10-70 kDa and pI 5.0-8.0. The identified proteins include mainly cytoplasmic and nuclear proteins and some mitochondrial, endoplasmic reticulum, and membrane proteins. These proteins are classified into different functional groups such as structure and mobility proteins (21.2%), metabolic enzymes (16.9%), protein folding and metabolism proteins (13.6%), signaling proteins (14.4%), heat-shock proteins (7.6%), and other functional proteins (12.7%). While structure and mobility proteins are mostly represented by protein spots with high abundance, signaling proteins are mostly represented by protein spots with relatively low abundance. Such a 2-DE database for RMC, especially with many signaling proteins and phosphoproteins characterized, will provide a valuable resource for comparative proteomics analysis of normal and pathologic conditions affecting MC function or pathologic progress.     

Phosphoproteome analysis of mouse liver using immobilized metal affinity purification and linear ion trap mass spectrometry

Since protein phosphorylation is a dominant mechanism of information transfer in cells, there is a great need for methods capable of accurately elucidating sites of phosphorylation. In recent years mass spectrometry has become an increasingly viable alternative to more traditional methods of phosphorylation analysis. The present study used immobilized metal affinity chromatography (IMAC) coupled with a linear ion trap mass spectrometer to analyze phosphorylated proteins in mouse liver. A total of 26 peptide sequences defining 26 sites of phosphorylation were determined. Although this number of identified phosphoproteins is not large, the approach is still of interest because a series of conservative criteria were adopted in data analysis. We note that, although the binding of non-phosphorylated peptides to the IMAC column was apparent, the improvements in high-speed scanning and quality of MS/MS spectra provided by the linear ion trap contributed to the phosphoprotein identification. Further analysis demonstrated that MS/MS/MS analysis was necessary to exclude the false-positive matches resulting from the MS/MS experiments, especially for multiphosphorylated peptides. The use of the linear ion trap considerably enabled exploitation of nanoflow-HPLC/MS/MS, and in addition MS/MS/MS has great potential in phosphoproteome research of relatively complex samples.     

蛋白质顺序测定技术(五)

(三)DABITC/PITC双偶合法~([1-13]) DABITC/PITC双偶合微量顺序测定法,是用有色试剂DABITC与多肽或蛋白质的氨基作第一次偶合,因该偶合反应产率只有25—50％左右,余下没有与DABITC偶合的氨基再用PITC作第二次偶合,由于PITC与多肽或蛋白质的偶合反应几乎是定量的,这样就保证了每个残基都反应完全,然后再裂解,转化、鉴定有色的DABTH-氨基酸。失去一个残基后的肽或蛋白质再偶合、裂解,转化,依次循环测定多肽和蛋白质顺序,其反应原理如下:     

Ab initio study of the spectroscopy of (CH3)(3)CN and (CH3)(2)CHN

Using the complete active space self-consistent field (CASSCF) method with 6-311++g(3df,3pd) basis sets, a few electronic states of nitrenes (CH3)3CN and (CH3)2CHN and their positive ions are calculated. All calculated states are valence states, and their characteristics are discussed in detail. In order to investigate the Jahn-Teller effect on (CH3)3CN radical, Cs symmetry was used for (CH3)3CN and (CH3)2CHN in the calculations. The results of our calculations (CASPT2 adiabatic excitation energies and RASSI oscillator strengths) suggest that the calculated transitions of (CH3)3CN at 27,710 cm(-1) and (CH3)2CHN at 28,110 cm(-1) are attributed to 23A' --> 13A', while those of (CH3)3CN at 28,916 cm(-1) and (CH3)2CHN at 29,316 cm(-1) are attributed to 13A' --> 13A'. The vertical and adiabatic ionization energies were obtained to compare with the photoelectron spectroscopic data. These results are in agreement with previous experimental data. Also, we present a comprehensive review on the CAS calculation results for (CH3)nCH(3-n)N (n = 0-3) presented in our previous and present papers.     

汽水混合换热器的实验研究和性能计算

换热器在工业和民用中有广泛应用。国务院节能指令要求,在规定期限内将蒸汽取暖改为热水取暖,因此如何改进换热器的性能、降低成本是当前一个有意义的问题。我们对QS系列汽水混合换热器进行了实验研究和理论分 ...     

中国电磁兼容认证委员会成立

中国电磁兼容认证委员会已于日前正式成立并开展电磁兼容认证工作。中国电磁兼容认证委员会是国家质量技术监督局依法授权的代表国家对产品电磁兼容性进行第三方公正评价的认证机构,由管理委员会和认证中心组成。中国电磁兼容认证委员会的工作受国家质量技术监督局领导,并接受社会各方面的监督。管理委员会由有关政府主管、生产、使用、科研和质量监督等部门的代表和专家组成。管理委员会负责制定委员会的章程、认证管理办法以及与认证有关的重要工作文件,领导和监督认证中心的工作。中国电磁兼容认证中心是管理委员会领导下的认证实体,负责…     

介绍一种新的压电材料——铌酸锂晶体

铌酸锂晶体是一种新的人工晶体,多用于激光、无线电等方面。由于它有良好的压电性能,也可以用作压力敏感元件。其最大的特点是Z切割的晶片有较小的侧向灵敏度,因而可以和目前大量应用的天然晶体电气石(Z切割)比美。在电气石货源不畅的情况下,人造晶体铌酸锂确实是一种较好的压电材料。      

裂缝介质渗流力学国内外科研方向调查(摘要)

前言遵照毛主席关于“按照实际情况决定工作方针,这是一切共产党员所必须牢牢记住的最基本的工作方法”和“调查就是解决问题”的教导,在探讨我国渗流力学科研方向时,我们结合我国油气田裂缝性的特点,调查了国外裂缝介质渗流力学科研现状和动向,并根据我国生产实际需要,写出了初步调查报告,现提要介绍如下。裂缝介质渗流力学是古典渗流力学中发展起来的一个分支,近年来得到迅速发展,其主要原因有二:      

晶体压杆传感器的动力特性

为了获得设计晶体压杆传感器的正确原则,本文对晶体压杆的动力性质进行了实验研究。研究的结果表明,就晶体与压杆的匹配问题而言,单纯考虑声阻抗匹配与否是不够的。为了取得正确的压力波形,就必须把晶体与压杆看作两个弹性振动系统,来研究它们的相互作用。根据这一分析,我们提出改善传感器性能的几种方法。经过实验,证明这些方法是有效的。