基于质谱技术蛋白质定量方法的研究进展

质谱技术已经成为目前蛋白质鉴定的重要工具。定量分析细胞内蛋白质组的动态变化,是当前研究蛋白质功能、揭示细胞生物机理、寻找疾病蛋白标记物和药物靶标的迫切需要。本文综述了基于质谱技术蛋白质定量的策略、方法和应用等方面近年来的进展,评述了几种蛋白质质谱定量方法的特点和应用潜力。     

Predictive Liquid Chromatography of Peptides Based on Hydrophilic Interactions for Mass Spectrometry-Based Proteomics

High-performance liquid chromatography (HPLC) is widely used for separation of complex peptide mixtures before mass spectrometry-based proteome analysis. In this analysis, reversed phase HPLC (RPHPLC) using non-polar stationary phases such as surface-modified silica containing alkyl groups (e.g., C18) is typically employed. Because of the high heterogeneity of proteomic samples, multidimensional separation approaches gained increasing attention recently to tackle this complexity and extremely high range of concentrations. In two-dimensional liquid chromatography, hydrophilic interaction chromatography (HILIC) is often a method of choice for combination with RP-HPLC because it uses reversed-phase type eluents and allows efficient separation of polar peptides. Due to the high degree of orthogonality in this two-dimensional separation space, it is tempting to develop approaches for predicting peptide retention times for HILIC-based separations similar to the ones for RP-HPLC. Recent successful efforts in this area were focused on developing retention coefficient (RC)-based approaches. Herein, we explored the feasibility of using a statistical thermodynamic model for prediction of peptide retention times in HILIC separations and determined the phenomenological parameters of the model for a bare silica column. The performance of the developed model was tested using HPLC-MS analysis of a set of synthetic peptides, as well as a tryptic peptide mixture.     

Analysis of Renal Cell Carcinoma as a First Step for Developing Mass Spectrometry-Based Diagnostics

Immediate diagnosis of human specimen is an essential prerequisites in medical routines. This study aimed to establish a novel cancer diagnostics system based on probe electrospray ionization-mass spectrometry (PESI-MS) combined with statistical data processing. PESI-MS uses a very fine acupuncture needle as a probe for sampling as well as for ionization. To demonstrate the applicability of PESI-MS for cancer diagnosis, we analyzed nine cases of clear cell renal cell carcinoma (ccRCC) by PESI-MS and processed the data by principal components analysis (PCA). Our system successfully delineated the differences in lipid composition between non-cancerous and cancerous regions. In this case, triacylglycerol (TAG) was reproducibly detected in the cancerous tissue of nine different individuals, the result being consistent with well-known profiles of ccRCC. Moreover, this system enabled us to detect the boundaries of cancerous regions based on the expression of TAG. These results strongly suggest that PESI-MS will be applicable to cancer diagnosis, especially when the number of data is augmented.     

SILAC-Pulse Proteolysis: A Mass Spectrometry-Based Method for Discovery and Cross-Validation in Proteome-Wide Studies of Ligand Binding

Reported here is the use of stable isotope labeling with amino acids in cell culture (SILAC) and pulse proteolysis (PP) for detection and quantitation of protein–ligand binding interactions on the proteomic scale. The incorporation of SILAC into PP enables the PP technique to be used for the unbiased detection and quantitation of protein–ligand binding interactions in complex biological mixtures (e.g., cell lysates) without the need for prefractionation. The SILAC-PP technique is demonstrated in two proof-of-principle experiments using proteins in a yeast cell lysate and two test ligands including a well-characterized drug, cyclosporine A (CsA), and a non-hydrolyzable adenosine triphosphate (ATP) analogue, adenylyl imidodiphosphate (AMP-PNP). The well-known tight-binding interaction between CsA and cyclophilin A was successfully detected and quantified in replicate analyses, and a total of 33 proteins from a yeast cell lysate were found to have AMP-PNP-induced stability changes. In control experiments, the method’s false positive rate of protein target discovery was found to be in the range of 2.1% to 3.6%. SILAC-PP and the previously reported stability of protein from rates of oxidation (SPROX) technique both report on the same thermodynamic properties of proteins and protein–ligand complexes. However, they employ different probes and mass spectrometry-based readouts. This creates the opportunity to cross-validate SPROX results with SILAC-PP results, and vice-versa. As part of this work, the SILAC-PP results obtained here were cross-validated with previously reported SPROX results on the same model systems to help differentiate true positives from false positives in the two experiments. Graphical Abstract

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A New Strategy of Using O18-Labeled Iodoacetic Acid for Mass Spectrometry-Based Protein Quantitation

A new O(18) labeling protocol is designed to assist quantitation of cysteine-containing proteins using LC/MS. Unlike other O(18) labeling strategies, the labeling is carried out at the intact protein level (prior to its digestion) during reduction/alkylation of cysteine side chains using O(18)-labeled iodoacetic acid (IAA). The latter can be easily prepared by exchanging carboxylic oxygen atoms of commercially available IAA in O(18)-enriched water at low pH. Since incorporation of the O(18) label in the protein occurs at the whole protein, rather than peptide level, the quantitation results are not peptide-dependent. The excellent stability of the label in mild pH conditions provides flexibility and robustness needed of sample processing steps following the labeling. In contrast to generally costly isotope labeling reagents, this approach uses only two relatively inexpensive commercially available reagents (IAA and H(2)O(18)). The feasibility of the new method is demonstrated using an 80?kDa human serum transferrin (hTf) as a model, where linear quantitation is achieved across a dynamic range spanning three orders of magnitude. The new approach can be used in quantitative proteomics applications and is particularly suitable for a variety of tasks in the biopharmaceutical sector, ranging from pharmacokinetic studies to quality control of protein therapeutics.     

False-Positive Rate Determination of Protein Target Discovery using a Covalent Modification- and Mass Spectrometry-Based Proteomics Platform

Detection and quantitation of protein–ligand binding interactions is important in many areas of biological research. Stability of proteins from rates of oxidation (SPROX) is an energetics-based technique for identifying the proteins targets of ligands in complex biological mixtures. Knowing the false-positive rate of protein target discovery in proteome-wide SPROX experiments is important for the correct interpretation of results. Reported here are the results of a control SPROX experiment in which chemical denaturation data is obtained on the proteins in two samples that originated from the same yeast lysate, as would be done in a typical SPROX experiment except that one sample would be spiked with the test ligand. False-positive rates of 1.2-2.2 % and <0.8 % are calculated for SPROX experiments using Q-TOF and Orbitrap mass spectrometer systems, respectively. Our results indicate that the false-positive rate is largely determined by random errors associated with the mass spectral analysis of the isobaric mass tag (e.g., iTRAQ®) reporter ions used for peptide quantitation. Our results also suggest that technical replicates can be used to effectively eliminate such false positives that result from this random error, as is demonstrated in a SPROX experiment to identify yeast protein targets of the drug, manassantin A. The impact of ion purity in the tandem mass spectral analyses and of background oxidation on the false-positive rate of protein target discovery using SPROX is also discussed.

Quantification of Protein-Ligand Interactions by Laser Electrospray Mass Spectrometry

Laser electrospray mass spectrometry (LEMS) measurement of the dissociation constant (K_d) for hen egg white lysozyme (HEWL) and N,N′,N″-triacetylchitotriose (NAG₃) revealed an apparent K_d value of 313.2?±?25.9 μM for the ligand titration method. Similar measurements for N,N′,N″,N″’-tetraacetylchitotetraose (NAG₄) revealed an apparent K_d of 249.3?±?13.6 μM. An electrospray ionization mass spectrometry (ESI-MS) experiment determined a K_d value of 9.8?±?0.6 μM. In a second LEMS approach, a calibrated measurement was used to determine a K_d value of 6.8?±?1.5 μM for NAG₃. The capture efficiency of LEMS was measured to be 3.6?±?1.8% and is defined as the fraction of LEMS sample detected after merging with the ESI plume. When the dilution is factored into the ligand titration measurement, the adjusted K_d value was 11.3 μM for NAG₃ and 9.0 μM for NAG₄. The calibration method for measuring K_d developed in this study can be applied to solutions containing unknown analyte concentrations.

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Graphical Abstract

     

Analysis of Pseudouridine by Fluorescence Spectrometry

Abstract

Fluorescence spectral properties of pseudouridine, an important biological indicator, have been measured for the first time in this study. The fluorescence quantum yield is maximal in aqueous solution with pH values above 9 with spectral properties characteristic of 5 alkyl substituted uracil derivatives. Chromatographic analysis shows the fluorescence is associated only with the pseudouridine peak. The concentration dependence of fluorescence is linear from 4 to 45 micromolar at pH 11.5. The excitation maximum is about 295 nm and the emission is broad with a maximum at about 390 nm. Addition of pseudouridine to urine extracts gives a linear increase in fluorescence with increasing pseudouridine concentration.     

Top-Down Analysis of Small Plasma Proteins Using an LTQ-Orbitrap. Potential for Mass Spectrometry-Based Clinical Assays for Transthyretin and Hemoglobin

Transthyretin (TTR) amyloidosis and hemoglobinopathies are the archetypes of molecular diseases where point mutation characterization is diagnostically critical. We have developed a Top-down analytical platform for variant and/or modified protein sequencing and are examining the feasibility of using this platform for the analysis of hemoglobin/TTR patient samples and evaluating the potential clinical applications. The platform is based on a commercial high resolution hybrid orbitrap mass spectrometer (LTQ-Orbitrap(?)) with automated sample introduction; automated data analysis is performed by our own software algorithm (BUPID topdown).The analytical strategy consists of iterative data capture, first recording a mass profile of the protein(s). The presence of a variant is revealed by a mass shift consistent with the amino acid substitution. Nozzle-skimmer dissociation (NSD) of the protein(s) yields a wide variety of sequence-defining fragment ions. The fragment ion containing the amino acid substitution or modification can be identified by searching for a peak exhibiting the mass shift observed in the protein mass profile. This fragment ion can then be selected for MS/MS analysis in the ion trap to yield sequence information permitting the identification of the variant. Substantial sequence coverage has been obtained in this manner. This strategy allows for a stepwise MS/MS analysis of the protein structure. The sequence information obtained can be supplemented with whole protein NSD fragmentation and MS/MS analysis of specific protein charge states. The analyses of variant forms of TTR and hemoglobin are presented to illustrate the potential of the method.     

A Quantitative Tool to Distinguish Isobaric Leucine and Isoleucine Residues for Mass Spectrometry-Based De Novo Monoclonal Antibody Sequencing

De novo sequencing by mass spectrometry (MS) allows for the determination of the complete amino acid (AA) sequence of a given protein based on the mass difference of detected ions from MS/MS fragmentation spectra. The technique relies on obtaining specific masses that can be attributed to characteristic theoretical masses of AAs. A major limitation of de novo sequencing by MS is the inability to distinguish between the isobaric residues leucine (Leu) and isoleucine (Ile). Incorrect identification of Ile as Leu or vice versa often results in loss of activity in recombinant antibodies. This functional ambiguity is commonly resolved with costly and time-consuming AA mutation and peptide sequencing experiments. Here, we describe a set of orthogonal biochemical protocols, which experimentally determine the identity of Ile or Leu residues in monoclonal antibodies (mAb) based on the selectivity that leucine aminopeptidase shows for n-terminal Leu residues and the cleavage preference for Leu by chymotrypsin. The resulting observations are combined with germline frequencies and incorporated into a logistic regression model, called Predictor for Xle Sites (PXleS) to provide a statistical likelihood for the identity of Leu at an ambiguous site. We demonstrate that PXleS can generate a probability for an Xle site in mAbs with 96% accuracy. The implementation of PXleS precludes the expression of several possible sequences and, therefore, reduces the overall time and resources required to go from spectra generation to a biologically active sequence for a mAb when an Ile or Leu residue is in question.

Quantification of Clenbuterol in Human Plasma and Urine by Liquid Chromatography-Tandem Mass Spectrometry

The quantitative determination of clenbuterol in human plasma and urine is of particular interest for various fields such as clinical and forensic research and doping controls. A simple and rapid sample preparation procedure based on liquid-liquid extraction with subsequent re-extraction followed by liquid chromatography and electrospray ionization tandem mass spectrometry allowed the determination of clenbuterol in urine and plasma at detection and quantification limits of 0.1 ng mL⁻¹ and 0.2 ng mL⁻¹, respectively, with recoveries ranging from 85–96%. The fast and robust nature of the assay provides a rapid and cost-effective alternative to established procedures utilizing solid-phase extraction strategies.     

高压液相色谱法测定肿瘤患者血清中假尿苷的含量

建立了血清中假尿苷含量的高效液相色谱测定法。结果表明，假尿苷在０３７５～１２μｍｏｌ／Ｌ范围内线性关系良好。最低检测量为００１５μｍｏｌ／Ｌ，回收率为９２７％，日内和日间相对标准偏差（ＲＳＤ）分别为４２％和４７％。用该法测定正常人及肝癌、肺癌、食管癌患者血清中假尿苷含量发现，３种肿瘤患者血清中假尿苷含量明显高于正常人（Ｐ＜００５），说明血清假尿苷可作为诊断恶性肿瘤的一种标志物。     

Quantification of Erdosteine in the Low-volume of Dog Plasma by Liquid Chromatography Tandem Mass Spectrometry

Introduction Erdosteine[N-(carboxymethylthioacetyl)-homocysteine thiolactone](Fig.1)is a synthetic derivative of natural amino acid methionine[1].It has been commonly used in clinical practice as a mucolytic drug and as an enhancer of respiratory ventilation in the treatment of patients with chronic obstructive respiratory disease.     

亲水相互作用色谱-串联质谱法快速测定葡萄酒中四种花青素

采用亲水相互作用色谱-串联质谱法(HILIC-MS/MS)建立同时测定葡萄酒中矢车菊素-3-O-葡萄糖苷、氯化葡萄糖苷芍药素、飞燕草素葡萄糖苷和氯化锦葵色素-3-O-葡糖苷4种花青素的分析方法。葡萄酒样品用甲醇直接稀释后,采用Merck ZIC HILIC色谱柱(150×2.1mm,3.5μm)分离;以乙腈-20mmol/L乙酸铵溶液作为流动相,梯度洗脱,目标化合物在多反应监测(MRM)模式下进行检测,外标法定量。在优化的条件下,4种花青素在1.0~50.0ng/mL范围内呈良好的线性关系,相关系数均不低于0.9992,方法定量限为0.05~1.0ng/mL。方法平均回收率为93.1%~96.7%,相对标准偏差不大于5.2%。该方法前处理简单、选择性好、灵敏度高,非常适用于葡萄酒中花青素的测定。     

Rapid and Sensitive Liquid Chromatography-Tandem Mass Spectrometry for Quantification of Glycyrrhetic Acid in Human Plasma

A simple, rapid and sensitive liquid chromatography-tandem mass spectrometry(LC-MS/MS) for the determination of glycyrrhetic acid in human plasma with ginsenoside Rh2 as internal standard was developed and validated. The plasma samples were prepared via liquid-liquid extraction with ethyl acetate. Chromatographic separation was accomplished on a Venusil MP-C18(50 mm×2.1 mm, 5 μm i.d.) column at 25 °C. The mobile phase consisted of acetonitrile/5 mmol?L-1 ammonium acetate(10:90, volume ratio) at a flow rate ...