专业英语文献翻译
班级：化学(师范)11—1
姓名：董文姣
学号：11055104
                         
Quantifying the Cluster of Differentiation 4 Receptor Density on Human T Lymphocytes Using Multiple Reaction Monitoring Mass Spectrometry。
reaction英语
ABSTRACT： Cluster of differentiation 4 （CD4） is an important glycoprotein containing four extracellular domains, a transmembrane portion and a short intracellular tail. It locates on the surface of various types of immune cells and performs a critical role in multiple cellular functions such as signal amplification and activation of T cells。 It is well-known as a clinical cell surface protein marker for study of HIV progression and for defining the T helper cell population in immunological applications。 Moreover， CD4 protein has been used as a biological calibrator for quantification of other surface and intracellular proteins. However， fl
ow cytometry, the conventional method of quantification of the CD4 density on the T cell surface depends on antibodies and has suffered from variables such as antibody clones, the ommatophore and conjugation chemistries, the fixation conditions， and the flow cytometric quantification methods used. In this study, we report the development of a highly reproducible na no liquid chromatography−multiple reaction monitoring mass spectrometry-based quantitative method to quantify the CD4 receptor density in units of copy number per cell on human CD4+ T cells. The method utilizes stable isotope—labeled full—length standard CD4 as an internal standard to measure
endogenous CD4 directly, without the use of antibodies. The development of the mass spectrometry-based approach of CD4 protein quantification is important as a complementary strategy to validate the analysis from the cytometry-based conventional method。 It also provides new support for quantitative understanding and advanced characterization of CD4 on CD4+ T cells。
Cluster of differentiation 4 （CD4） is a glycoprotein that locates on the surface of immune
cells such as T helper cells, monocytes, macrophages， and dendritic cells. As a co receptor， CD4 amplifies the signal generated by the T cell receptor, which is essential for activation of many molecules involved in the signaling cascade of an activated T cell. In human T lymphocytes, CD4 receptor protein is encoded by the CD4 gene1and has four distinct extracellular domains （D1 to D4）, a transmembrane portion, and a short intracellular tail。2The use of antihuman CD4 monoclonal antibodies generated against the four extracellular domains has been widely used to define T helper cells in phenotypical. Although the number of CD4+ T cells decreases in the progression of HIV-1 viral infection deriving from the gp120 viral protein binding to the CD4 receptor， Ponce let et al. reported that the surface CD4 density still remained constant on T helper cells of HIV-1 infected individuals。3Since then, multiyear research has supported the theory that CD4 expression/density can be used as a biological calibrator for quantification of other surface
and intracellular proteins.4
Quantitative multicolor flow cytometry incorporating anti— bodies and a fluorescence detec
tion method has played a critical role in clinical diagnostics and immunotherapies. Though the ultimate objective of quantitative flow cytometry is to measure the number of antigens or ligand binding sites associated with a cell， the task is carried out by measuring the number of antibodies bound per cell (ABC）。 It is critically important to produce biological cell reference materials that bear well-characterized protein markers such as CD4 for the trans—formation of a calibrated linear fluorescence intensity scale of a flow cytometer channel to a biologically meaningful ABC scale。7The quality of the cytometric measurements is affected by variables such as antibody clones, the ommatophore and conjugation chemistries, the fixation conditions， and the flow cytometric quantification methods used.4,8−11Hence, in addition to characterizing candidate reference cell preparations that use antibody-based cytometric methods，12it is necessary to develop a complementary approach to validate the absolute quantification of reference marker proteins such as CD4 without the use of antibodies.
Liquid chromatography coupled mass spectrometry has emerged as a versatile platform for quantitative protein/proteges analysis due to its high specificity and sensitivity。 Relative q
uantification of proteins can be achieved without the use of any internal standard for comparative analysis under the same catalytical conditions。 However， in many analyses such as clinical biomarker tests, absolute quantification of protein（s) in terms of molecule copy number per cell or per unit weight/volume of biological samples is required。Absolute quantitative data enable valuable comparisons across different studies and conclusive interpretations of the disease states or treatment efficacy as well as the understanding of the whole body system biology probed from different angles in different studies。 Multiple reaction monitoring mass spectrometry （MRM MS） combining proper separation and/or fractionation techniques has been proven to be an effective platform for protein quantification in biological samples。16−18In the present study， we report the development of an MRM MS-based approach that combines scalepan liquid chromatography and a stable isotope—labeled full—length protein as the internal standard， enabling the quantification of the CD4 receptor density in units of copy number per cell on human CD4+ T cells without the use of antibodies。
EXPERIMENTAL SECTION
Materials. All chemicals and reagents， unless indicated specifically, were from Sigma—Ald rich Inc。
Determination of the Human CD4+ T Cell Count。Cryopreserved, negatively selected human CD4+ T cells with a purity of 98.5％ were purchased from Astarte Biologics
(Redmond, WA), confirmed internally, and used without further purification。 The thawed cryopreserved CD4+ T cells were slowly added to 9 L of RPMI—1640 containing 10％ fetal bovine serum （FBS) in a 15 L conical tube。 After the tube was inverted three times, the cells were centrifuged at 400gnfor 10 min, and the supernatant was discarded。 The resulting cells were washed once and re suspended in phosphate—buffered saline (PBS） with 1％ FBS. The number of CD4+ T cells was counted by using both a hemocytometer and a flow cytometry with which Count beads from BD Bioscience (San Jose,CA） were used as the internal counting standard。 Mouse antihuman CD4 fluorescein isocyanate （FITC； clone SK3，catalog number 340133, BD Biosciences） was used for cell staining， and CD4+ cells were counted using an Aria II flow sorter from BD Biosciences.
Gating of CD4+ and Count beads was performed on a FITC histogram. The ratio of the respectively gated events of CD4+ cells and Count beads was used for obtaining the CD4+ cell number according to the manufacturer’s procedure。 The CD4+ cell numbers measured by the hemocytometer and flow cytometry were fairly consistent with a difference of no more than 6%， and therefore, the averaged cell count from both methods was used to derive the CD4 receptor density/copy number per cell.

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