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Chinese Journal of Chromatography
2021, Vol. 39, No. 1
Online: 08 January 2021

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Volume 39, Number 1 Content 2021, 39 (1):  0-0.  Abstract ( 45 )   PDF (3836KB) ( 81 )

Communications

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Recent advances in sample pretreatment techniques for chromatographic analysis HUANG Weini, LIN Zian 2021, 39 (1):  1-3.  DOI: 10.3724/SP.J.1123.2020.05011 Abstract ( 238 )   HTML ( 532 )   PDF (832KB) ( 233 )

Mini-reviews

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Recent advances in method development and application of multi-collector inductively coupled plasma mass spectrometry ZHANG Luyao, CHEN Zigu, YANG Xuezhi, LU Dawei, LIU Qian, JIANG Guibin 2021, 39 (1):  4-9.  DOI: 10.3724/SP.J.1123.2020.07030 Abstract ( 268 )   HTML ( 48 )   PDF (1008KB) ( 138 )   Stable isotopic analysis is an important branch of analytical chemistry. Accurate determination of the stable isotopic compositions of substances is critical for tracing their sources and investigating their transformation processes. The new generation of mass spectrometry technology has greatly facilitated the development of high-precision stable isotopic analysis methods. In particular, multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS) has emerged as a powerful tool for isotopic composition determination. However, isotopic analysis by MC-ICP-MS is highly sensitive to the sample matrix, which may compromise the precision and accuracy of the analysis. Therefore, it is particularly important to reduce the sample matrix effect using efficient sample purification techniques. This article summarizes the recent progress in sample purification and instrument hyphenation methods for MC-ICP-MS, and provides perspectives on the future application of MC-ICP-MS in different fields.

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Effects of peak compression in gradient elution of liquid chromatography HAO Weiqiang, LIU Lijuan, SHEN Qiaoyin 2021, 39 (1):  10-14.  DOI: 10.3724/SP.J.1123.2020.07042 Abstract ( 179 )   HTML ( 65 )   PDF (960KB) ( 156 )   Peak compression is a unique feature of gradient elution and is non-existent in isocratic elution. Since the classical plate height equation, which is also called as van Deemter equation, is derived by assuming isocratic elution, it cannot be used to account for the effects of peak compression. As opposed to the case of isocratic elution, the retention factor (k) varies with the mobile phase composition (φ) under gradient elution, thereby complicating mathematical analysis. Herein, the research progress on peak compression in the past decade, especially the effect of the nonlinear feature of solvent strength models (i. e., expressions for ln k vs. φ), is reviewed. A general expression for the peak compression factor (G) is introduced, for which the variation in plate height (H) with φ is ignored. Based on this equation, the classical equation for G, which was first proposed by Poppe and assumes the linear solvent strength model (LSSM) and linear gradient elution, can be derived. The effects of pre-elution of the solute in the initial mobile phase on G, which are attributed to the dwelling time of the system, are included in the Poppe equation. When the solvent strength model is nonlinear, e. g., the quadratic solvent strength model (QSSM), the analytical expressions for G can also be obtained from the general expression. Under ideal chromatographic conditions, where H=0 and the adsorption isotherm is linear, the peak compression is determined by the ratio of the retention factor of the solute in the initial mobile phase to that at the eluted mobile phase composition.

Reviews

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Advances in enrichment of phosphorylated peptides and glycopeptides by smart polymer-based materials ZHENG Xintong, WANG Xue, ZHANG Fusheng, ZHANG Xuyang, ZHAO Yanyan, QING Guangyan 2021, 39 (1):  15-25.  DOI: 10.3724/SP.J.1123.2020.05036 Abstract ( 165 )   HTML ( 45 )   PDF (3421KB) ( 93 )   Protein post-translational modification (PTM) is at the forefront of focus of proteomics research. It not only regulates protein folding, state, activity, localization, and protein interactions, but also helps scientists understand the biological processes of organisms more comprehensively, providing stronger support and basis for the prediction, diagnosis, and treatment of diseases. In living organisms, there are more than 300 types of PTMs of proteins and their modification processes are dynamic. At the same time, protein modifications do not exist in isolation. The occurrence of the same physiological or pathological process requires the joint action of various modified proteins, which affect and coordinate with each other. Owing to the low abundance of PTM products (e. g., phosphorylated peptides or glycopeptides) and the presence of strong background interference, it is difficult to analyze them directly through mass spectrometry. Therefore, the development efficient materials and techniques for the selective enrichment of PTM peptides is urgently needed. Conventional separation methods have partially solved the challenges involved in the enrichment of glycopeptides and phosphorylated peptides; however, there are some inevitable issues, such as the excessive binding force of metal ions (e. g., Fe3+and Ti4+) toward multiple phosphorylated peptides, resulting in difficulty in elution and identification through mass spectrometry. In addition, owing to the insufficient binding affinity of materials toward glycopeptides, most glycopeptides that have been identified at present are of the sialic acid type, and a large number of neutral glycans, for instance, O-link glycopeptides and high mannose-type glycans are difficult to enrich and identify. The emergence of smart polymers provides a new avenue for the development of PTM-enriched materials. Several studies have reported that smart polymers can reversibly change their structure and function through external physical, chemical, or biological stimulation, to achieve highly controllable adsorption and desorption of phosphorylated peptides and glycopeptides. Based on this strategy, a series of novel enrichment materials and methods have been developed, which have greatly attracted the interest of researchers. On the one hand, the response changes of smart polymers include the increase or decrease of hydrophobicity, the change of shape and morphology, the redistribution of surface charge, the exposure or hiding of affinity ligands, etc. Changes in these properties can be achieved by simply changing external conditions such as temperature, pH, solvent polarity, and biomolecules. These properties, in turn, enable the fine-tuning of the affinity between the target and the smart polymers. Furthermore, the affinity can provide an additional driving force, which can significantly improve biological separation. On the other hand, smart polymers provide a series of convenient and expandable platforms for integrating various functional modules, such as specific recognition components, which will facilitate the development of novel enrichment materials for protein methylation, acetylation, and ubiquitination. Smart polymer materials show great potential in the field of separation, which is promising for the analysis and research of protein PTMs. This review summarizes the research progress of smart polymer materials for the separation and enrichment of phosphorylated peptides and glycopeptides according to nearly 50 representative articles from the Web of Science in the past two decades.

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Advances in the application of affinity separation for analyzing protein ubiquitination ZHONG Huifei, HUANG Yanyan, JIN Yulong, ZHAO Rui 2021, 39 (1):  26-33.  DOI: 10.3724/SP.J.1123.2020.07005 Abstract ( 203 )   HTML ( 29 )   PDF (2541KB) ( 157 )   Protein ubiquitination is one of the most common yet complex post-translational modifications in eukaryotes that plays an important role in various biological processes including cell signal transduction, growth, and metabolism. Disorders in the ubiquitination process have been revealed to correlate with the occurrence and development of many diseases such as neurodegenerative disease, inflammation, and cancer. Investigation of protein ubiquitination is of great importance to uncover protein functions, understand the molecular mechanisms underlying biological processes, and develop novel strategies for disease treatment. Great advances have been made toward understanding protein ubiquitination; however, it remains a challenging task due to the high diversity of ubiquitination sites and structures, as well as the dynamic nature of ubiquitination in biological processes. Protein ubiquitination occurs through the formation of a covalent bond between the carboxyl terminus of ubiquitin and the ε-amino group of a lysine residue in the substrate. As a small protein, ubiquitin itself can be further modified by another ubiquitin molecule to form homotypic or heterotypic polyubiquitin chains. There are eight sites, namely seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) and one N-terminal methionine (M1), in one ubiquitin molecule that can be used to form a ubiquitin dimer. The variations in modification sites, ubiquitin chain lengths, and conformations result in differences in protein sorting, cell signaling, and function. To resolve the high complexity of protein ubiquitination, new separation approaches are required. Affinity separation based on the specific recognition between biomolecules offers high selectivity and has been employed to study the structures and functions of ubiquitination. In addition, affinity ligands are central to the separation performance. Different affinity ligands have been developed and employed for the capture and enrichment of ubiquitylated proteins. Immunoaffinity separation based on antigen-antibody interactions has been one of the most classical separation methods. Antibodies against ubiquitin or different ubiquitin linkages have been developed and widely applied for the enrichment of ubiquitylated proteins or peptides. The specific capture allows the downstream identification of endogenous ubiquitination sites via mass spectrometry and thus facilitates understanding of the roles and dynamics of polyubiquitin signals. Ubiquitin-binding domains (UBDs) are a collection of modular protein domains that can interact with ubiquitin or polyubiquitin chains. Ubiquitin-associated domains, ubiquitin-interacting motifs, and ubiquitin-binding zinc finger domains are the most frequently used UBDs. Due to the moderate affinity of UBDs toward ubiquitin or ubiquitin chains, tandem ubiquitin-binding entities (TUBEs) have been engineered with high affinities (Kd in the nanomolar range) and exhibit potential as powerful tools for ubiquitination analysis. Because of their affinity and selectivity, UBDs and TUBEs have been applied for the isolation and identification of ubiquitylated targets in cancer cells and yeasts. Compared with antibodies and UBDs, peptides are smaller in size and can be facilely synthesized via chemical approaches. The modular structure of peptides allows for de novo design and screening of artificial ubiquitin affinity ligands for targeted capture of ubiquitinated proteins. Furthermore, the polyhistidine tag at the N-terminus of ubiquitin facilitates the purification of ubiquitylated substrates using immobilized metal affinity chromatography. Considering the high complexity of biosystems, strategies combining multiple affinity ligands have emerged to further improve separation efficiency and reduce background interference. Several combinations of antibodies with UBDs, antibodies with peptidyl tags, and UBDs with peptidyl tags have been developed and proven to be effective for the analysis of protein ubiquitination. These affinity-based approaches serve as important solutions for studying the structure-activity relationship of protein ubiquitination. This review highlights the applications and recent advances in affinity separation techniques for analyzing protein ubiquitination, focusing on the methods using antibodies, UBDs, peptides, and their combinations as affinity ligands. Further, their applications in the enrichment of ubiquitin-modified substrates and the identification of ubiquitination structures are introduced. Additionally, remaining challenges in affinity separation of protein ubiquitination and perspectives are discussed.

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Recent advances in sample preparation technologies for analysis of harmful substances in aquatic products WANG Xingyi, CHEN Yanlong, XIAO Xiaohua, LI Gongke 2021, 39 (1):  34-45.  DOI: 10.3724/SP.J.1123.2020.07025 Abstract ( 207 )   HTML ( 30 )   PDF (2433KB) ( 145 )   Aquatic products, which are among the most important sources of animal protein, contain proteins, vitamins, and a variety of trace elements, thus occupying an indispensable part of a reasonable diet. China is the largest consumer market of aquatic products in the world. The quality and safety of aquatic products are closely related not only to the healthy development of the aquaculture industry, but also to people’s health. However, the presence of harmful substances has a bearing on the quality and safety of aquatic products in the overall process, including breeding, processing, storage, and transportation. These harmful substances are enriched in aquatic products and are transferred to humans via the food chain. Accurate determination of such harmful substances in aquatic product samples is imperative because of their complex matrices and extremely low concentrations. Many efficient sample preparation techniques such as liquid-liquid extraction, solid-phase extraction, and QuEChERS (quick, easy, cheap, effective, rugged, and safe method) with different configurations have been developed and widely employed for preconcentration in different matrices of aquatic products. Meanwhile, solid-phase microextraction has been demonstrated to be advantageous for some volatile and ultra-trace harmful substances. Suitable sample preparation techniques are important for effectively removing matrix interferences as well as for improving the sensitivity and accuracy of the method. It is important to develop appropriate sample preparation techniques for different target compounds in aquatic products. The harmful substances in aquatic products can be segregated into three categories according to their sources: (1) environmental pollutants in aquatic products; (2) substances acquired during aquaculture, transportation, and processing; (3) biotoxins in aquatic products. This article reviews the progress in sample pretreatment techniques for three harmful substances in aquatic products over the past decade. Various sample pretreatment techniques have been summarized and described, including liquid-liquid extraction, solid-phase extraction, solid-phase microextraction, QuEChERS, and magnetic solid-phase extraction. In addition, the merits and demerits of these techniques and future research directions are discussed. Finally, we reviewed the progress in functionalized materials for the preparation of aquatic product samples. With the increasing demand for aquatic products, quick, sensitive, and practical detection methods, such as surface-enhanced Raman scattering (SERS) are gaining importance. SERS has great potential for fast and accurate on-site detection of harmful substances in aquatic products. Several nondestructive sample pretreatment techniques have also been developed for harmful substances in aquatic products. The application and development of these techniques will guarantee the safety of aquatic products. Moreover, in vivo solid-phase microextraction is a potential method for aquatic product analysis. This technique integrates sampling, extraction, and enrichment into a single step, thus significantly reducing the processing time, labor, and cost. Overall, with the development and application of sophisticated materials and techniques, we can expect theoretical and practical advances in aquatic product analysis.

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Research progress on analytical methods for the determination of hexachlorobutadiene WANG Yaotian, ZHANG Haiyan, SHI Jianbo, JIANG Guibin 2021, 39 (1):  46-56.  DOI: 10.3724/SP.J.1123.2020.05019 Abstract ( 326 )   HTML ( 32 )   PDF (1704KB) ( 206 )   Hexachlorobutadiene (HCBD) is one of persistent organic pollutants (POPs) listed in Annex A and Annex C of the Stockholm Convention in 2015 and 2017, respectively. Research on the sources, environmental occurrences, and biological effects of HCBD has a great significance in controlling this newly added POPs. Sensitive and credible methods for the determination of HCBD are preconditions and form the basis for related research work. In recent years, many researchers have included HCBD as one of the analytes in monitoring or methodological studies. Based on the results of these studies, this paper reviews the research progress on analytical methods for the determination of HCBD and focuses on sample pretreatment methods for the analysis of HCBD in various matrices such as air, water, soil, sewage sludge, and biological tissues. The advantages and disadvantages of the methods are also compared to provide reference for further research in this field. For air samples, HCBD was usually collected by passing air through sorbent cartridges. Materials such as Tenax-TA, Carbosieve, Carbopack, Carboxen 1000, or their mixtures were used as the sorbent. HCBD was thermally desorbed and re-concentrated in a trap and finally transferred for instrumental analysis. Limits of detection (LODs) for HCBD in these methods were at the ng/m3 scale. Compared to sampling using pumps, passive air samplers (PAS) such as polyurethane foam PAS (PUF-PAS) do not require external power supply and are more convenient for sampling POPs in air at a large scale. The LOD of the sorbent-impregnated PUF PAS (SIP-PAS) method was much lower (0.03 pg/m3) than that of the PUF-PAS method (20 pg/m3). However, the sampling volumes in the SIP-PAS and PUF-PAS methods (-6 m3) calculated from the log KOA value of HCBD have significant uncertainty, and this must be confirmed in the future. For water samples, HCl or copper sulfate was added to the sample immediately after sampling to prevent any biological activities. HCBD can be extracted from water using methods such as the purge and trap method, liquid-liquid extraction (LLE) method, and solid phase extraction (SPE) method. Among these methods, SPE enabled the simultaneous extraction, purification, and concentration of trace HCBD in a single step. Recoveries of HCBD on Strata-X and Envi-Carb SPE cartridges (63%-64%) were higher than those on Envi-disk, Oasis HLB, and Strata-C18 cartridges (31%-46%). Drying is another key step for obtaining high recoveries of HCBD. Disk SPE involving the combination of a high-vacuum pump and a low-humidity atmosphere is an effective way to eliminate the residual water. In addition, a micro SPE method using functionalized polysulfone membranes as sorbents and employing ultrasonic desorption was developed for extracting HCBD from drinking water. The recovery of HCBD reached 102%, with a relative standard deviation (RSD) of 3.5%. For solid samples such as dust, soil, sediment, sewage sludge, fly ash, and biota tissue, multiple pretreatment methods were used in combination, owing to the more complex matrix. Freeze or air drying, grinding, and sieving of samples were commonly carried out before the extraction. Soxhlet extraction is a typical extraction method for HCBD; however, it requires many organic reagents and is time consuming. The accelerated solvent extraction (ASE) method requires a small amount of organic reagent, and the extraction can be performed rapidly. It was recently applied for the extraction of HCBD from solid samples under 10.34 MPa and at 100 ℃. Purification could be achieved simultaneously by mixing florisil materials with samples in the ASE pool. Nevertheless, employing the ASE methods widely is difficult because of their high costs. Ultrasonic-assisted extraction (UAE) has the same extraction efficiency for HCBD, with much lower costs compared to ASE, and is therefore adopted by most researchers. The type of extraction solvent, solid-to-liquid ratio, ultrasonic temperature, and power affect the extraction efficiency. Ultrasonic extraction at 30 ℃ and 200 W using 30 mL dichloromethane as the extraction solvent resulted in acceptable recoveries (64.0%-69.4%) of HCBD in 2 g fly ash. After extraction, a clean-up step is necessary for the extracts of solid samples. Column chromatography is frequently used for purification. The combined use of several columns or a multilayer column filled with florisil, silica gel, acid silica gel, or alumina can improve the elimination efficiency of interfering substances. Instrumental analysis for HCBD is mainly performed with a gas chromatograph equipped with a mass spectrometer operating in selected ion monitoring mode. DB-5MS, HP-5MS, HP-1, ZB-5MS, and BP-5 can be used as the chromatographic columns. Qualification ions and quantification ions include m/z 225, 223, 260, 227, 190, and 188. GC-MS using an electron ionization (EI) source was more sensitive to HCBD than GC-MS using a positive chemical ionization source (PCI) and atmospheric pressure chemical ionization source (APCI). Gas chromatography-tandem mass spectrometry (GC-MS/MS), gas chromatography-high-resolution mass spectrometry (GC-HRMS), and high-resolution gas chromatography-high-resolution mass spectrometry (HRGC-HRMS) have recently been used for the separation and determination of HCBD and various other organic pollutants. Instrumental detection limits for HCBD in GC-MS/MS, GC-HRMS, and HRGC-HRMS were more than ten times lower than that in GC-MS, indicating the remarkable application potential of these high-performance instruments in HCBD analysis.

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Application of gas chromatography separation based on metal-organic framework material as stationary phase TANG Wenqi, MENG Shasha, XU Ming, GU Zhiyuan 2021, 39 (1):  57-68.  DOI: 10.3724/SP.J.1123.2020.06028 Abstract ( 234 )   HTML ( 30 )   PDF (3362KB) ( 237 )   Metal-organic frameworks (MOFs) are a new class of porous materials, which are synthesized using organic ligands and inorganic metal ions or metal clusters. MOFs possess tunable structures through the self-assembly of a large number of organic linkers and metal nodes, which is beyond the scope of conventional porous materials. In addition, MOFs have excellent properties, including the lowest density (as low as 0.13 g/cm), highest specific surface area (as high as 10400 m2/g), and largest pore aperture (as large as 9.8 nm) among all porous materials reported till date. Because of their high porosity, large surface area, tunable apertures, as well as high chemical and thermal stabilities, MOFs have been widely applied in the fields of adsorption, separation, and catalysis. In addition, MOFs have been successfully applied as stationary phases for isomer separation in gas chromatography (GC). Since the use of the first MOF (MOF-508) packed column for the separation of alkane isomers in GC, several other MOFs (e. g., MIL-47, MOF-5, and ZIF-8) have been employed for the GC separation of isomers. However, packed-column-type separation not only requires gram-scale quantities of MOFs, thereby increasing the analysis cost, but also results in poor separation efficiency. The first MOF (MIL-101) capillary column designed toward cost reduction allowed for the baseline separation of xylene and ethylbenzene isomers within 100 s under constant-temperature conditions. Since then, the capillary-type column has been widely utilized in the MOF-based stationary phase for GC separation. Alkanes, xylene isomers and ethyl toluene, oxy-organics and organic pollutants are not only important chemicals in industry but also harmful environmental pollutants. Thus, the separation of these analytes is of practical importance environmental monitoring and industrial quality control. However, it is difficult to realize the efficient separation and detection of these isomers or racemates because of their similar boiling points and molecular sizes. In the past decades, GC was utilized as a rapid and efficient technique for the separation of the abovementioned analytes. The stationary phase used in GC plays a dominant role in the separation processes. This review summarizes the MOF-based GC separation of the abovementioned targets based on the different classification of analytes, including alkanes, xylenes, racemates, oxy-organics and persistent organic pollutants. The separation mechanisms of different analytes are also discussed according to the structural benefits of MOFs. The separation mechanisms mainly involve van der Waals forces between the MOFs and analytes, interactions between the unsaturated metal sites and different functional groups of the analytes, molecular sieve effect or shape selectivity, and hydrogen-bond or π-π interactions. In addition, the chiral recognition abilities of MOFs possibly depend on the interactions between the chiral active sites in chiral MOFs and racemates. Furthermore, efficient GC separation is influenced by thermodynamic and kinetic factors. The thermodynamic factor is mainly the difference between the partition coefficients of the separated components, which also reflects the properties of the analytes as well as the interactions between the stationary phase and the analytes. The kinetic factor also affects the column efficiency and chromatographic peak shape. Compared with traditional inorganic porous materials, MOFs with tunable structures are more favorable for optimizing the separation of isomers from both thermodynamic and kinetic standpoints. Therefore, this review summarizes the separation mechanism when using MOFs as stationary phases for isomer separation via thermodynamic and kinetic analyses. We hope the review would aid the state-of-art design of MOF stationary phases for high efficient isomer separations in GC.

Articles

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Determination of 16 organophosphate esters in human blood by high performance liquid chromatography-tandem mass spectrometry combined with liquid-liquid extraction and solid phase extraction HOU Minmin, SHI Yali, CAI Yaqi 2021, 39 (1):  69-76.  DOI: 10.3724/SP.J.1123.2020.07033 Abstract ( 205 )   HTML ( 49 )   PDF (1406KB) ( 173 )   Measurement of organophosphate esters (OPEs) in human body fluids is important for understanding human internal exposure to OPEs and for assessing related health risks. Most of the current studies have focused on the determination of OPE metabolites in human urine, as OPEs are readily metabolized into their diester or hydroxylated forms in the human body. However, given the existence of one metabolite across multiple OPEs or multiple metabolites of one OPE, as well as the low metabolic rates of several OPEs in in vitro studies, the reliability of urinary OPE metabolites as biomarkers for specific OPEs is needs to be treated with caution.Human blood is a matrix that is in contact with all body organs and tissues, and the blood levels of compounds may better represent the doses that reach target tissues. Currently, only a few studies have investigated the occurrence of OPEs in human blood by different analytical methods, and the variety of OPEs considered is limited. In this study, a method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous determination of 16 OPEs in human blood, and the extraction efficiency of the solid phase extraction (SPE) column for OPEs was verified. To human blood samples, 10 ng of an internal standard was added, followed by mixing and aging for 30 min. The samples were extracted three times with acetonitrile using a shaker, and then purified on ENVI-18 cartridges with acetonitrile containing 25% dichloromethane as the eluent. Finally, the OPEs were analyzed by high performance liquid chromatography-tandem mass spectrometry. After optimization of the analytical column and mobile phases, the analytes were separated on a BEH C18 column (100 mm×2.1 mm, 1.7 μm) by gradient elution using methanol and 5 mmol/L ammonium acetate in water as the mobile phase. Then, the analytes were ionized in electrospray ionization positive (ESI+) mode and detected in the multiple reaction monitoring (MRM) mode. The mass spectral parameters, including the precursor ion, product ion, declustering potential, entrance potential, and collision cell exit potential, were optimized. The results were quantified by the internal standard method. The limits of detection (LOD, S/N=3) of the OPEs were in the range of 0.0038-0.882 ng/mL. The calibration curves for the 16 OPEs showed good linear relationships in the range of 0.1-50 ng/mL, and the correlation coefficients were >0.995. The extraction efficiency of the ENVI-18 column for the 16 OPEs was validated, and the average recoveries of the target compounds were 54.6%-104%. The average recoveries (n=3) of 15 OPEs, except trimethyl phosphate (TMP), in whole blood at three spiked levels were in the range of 53.1%-126%, and the relative standard deviations (RSDs) were in the range of 0.15%-12.6%. The average recoveries of six internal standards were in the range of 66.8%-91.6% except for TMP-d9 (39.1%), with RSDs of 3.52%-6.85%. The average matrix effects of the OPEs in whole blood were 56.4%-103.0%. Significant matrix effects were found for resorcinol bis(diphenyl phosphate) (RDP) (75.8%±1.4%), trimethylphenyl phosphate (TMPP) (68.4%±1.0%), 2-ethylhexyl di-phenyl phosphate (EHDPP) (56.4%±12.4%), and bisphenol-A bis(diphenyl phosphate) (BABP) (58.5%±0.4%). However, these effects could be corrected by similar signal suppressions of the corresponding internal standard (TPHP-d15, 77.4%±7.5%). This method is simple, highly sensitive, and suitable for the determination of OPEs in human blood. Fifteen human whole blood samples were collected to quantify the 16 OPEs using the developed method. The total concentrations of the OPEs ranged from 1.50 to 7.99 ng/mL. The detection frequencies of eight OPEs were higher than 50%. Tri-iso-butyl phosphate (TiBP), tri(2-chloroethyl) phosphate (TCEP), and tri(1-chloro-2-propyl) phosphate (TCIPP) were the dominant OPEs, with median concentrations of 0.813, 0.764, and 0.690 ng/mL, respectively. These results indicated widespread human exposure to OPEs, which should be of concern.

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Affinity chromatography based phosphoproteome research on lung cancer cells and its application ZHANG Baohui, WANG Chentong, GUO Miao, XIAO Hua 2021, 39 (1):  77-86.  DOI: 10.3724/SP.J.1123.2020.07041 Abstract ( 137 )   HTML ( 38 )   PDF (1700KB) ( 68 )   Phosphorylation is one of the most important post-translational modifications in proteins. It plays a key role in numerous cellular processes, including signal transduction, cell proliferation, and intercellular communication. More than 30% of the cellular proteins are phosphorylated at a given time. However, dysregulation of phosphorylated proteins usually leads to a disorder in the intracellular signaling pathways and the onset of various diseases, especially cancer. Cell proliferation and metastasis are the major manifestations of cancer progression, and these might be affected by the protein phosphorylation levels. Clinically, cancer usually metastasizes at the middle and late stages, affecting other organs beyond primary lesion. This poses significant challenges in cancer treatment and prognosis. Consequently, comparing the phosphorylated proteomes of cells with different metastatic capabilities is helpful in studying the role of protein phosphorylation in cancer metastasis and progression. The human low metastatic lung cancer cell line 95C and high metastatic lung cancer cell line 95D are two of the four sublines isolated from human lung giant cell carcinoma cell line (PLA-801) by the single-cell cloning technique. These are ideal models for studying tumor metastasis and non-small cell lung cancer. MRC-5 cell line was obtained from a 14 week old fetal normal lung tissue. Quantitative analysis of the proteome and phosphorylated proteome in these normal lung cells and lung cancer cells with different metastatic capacities can identify key pathways and regulatory proteins associated with lung cancer metastasis and progression. Immobilized metal affinity chromatography (IMAC) is an efficient technique for the enrichment of phosphopeptides and has been widely used for phosphoproteome research. Metal ions (such as Ti4+) are immobilized on the substrate by chelation, and phosphopeptides can be selectively adsorbed under acidic conditions and eluted under alkaline conditions. IMAC can enrich phosphate groups at different amino acid sites with high specificity. In this study, Ti4+was chelated onto Ti4+-IMAC material, which was used to enrich phosphopeptides for phosphoproteome research. Two enrichment methods, namely, the vortexing method and solid phase extraction (SPE) method, were first compared for the enrichment of phosphopeptides using 10 μm Ti4+-IMAC. Phosphopeptides were highly enriched using the vortexing method. Following this, two sizes of Ti4+-IMAC material (10 μm and 30 μm) were compared to determine the efficiency of phosphopeptide enrichment. Enrichment efficiency was superior with the smaller-sized material. Therefore, the small-size Ti4+-IMAC material was selected for the proteomics research of lung cell phosphorylation. The optimized strategy was further used to compare the phosphoproteomes of the lung cancer cells with different metastatic abilities. Label-free quantification proteomics demonstrated that 510, 863, and 1108 phosphorylated proteins were identified from normal lung fibroblasts (MRC-5), low metastatic lung cancer cells (95C), and high metastatic lung cancer cells (95D), respectively, using the optimized Ti4+-IMAC method. Among them, 317 phosphorylated proteins were shared among the three groups. The protein phosphorylation level increased significantly with increasing cellular metastatic capacity. In our study, 7560 phosphorylation sites were identified on 1268 phosphorylated proteins, among which 1130 phosphorylation sites were differentially expressed. Some abnormally expressed kinases and their phosphorylation levels are closely associated with malignant cell proliferation. Comparative bioinformatics analysis showed that dysregulated phosphoproteins were mainly related to cell migration functions, such as cell invasion, migration, and death. These abnormally expressed phosphorylated proteins and phosphorylation sites could be further validated and studied for lung cancer metastasis. Our study demonstrates that Ti4+-IMAC is a powerful tool for conducting cancer metastasis-related phosphoproteome research. By optimizing the phosphopeptide enrichment strategy, our data preliminarily clarified the correlation between the abnormality of the phosphoprotein network and lung cancer metastasis. This is expected to be useful for studying phosphorylation sites, phosphorylated proteins, and their signaling pathways related to lung cancer progression.

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Size exclusion-reverse liquid column chromatography-mass spectrometry and its application in the identification of post-translationally modified proteins in rat kidney LI Jianmin, ZHUO Yue, ZHANG Yida, LI Na, WU Jianlin 2021, 39 (1):  87-95.  DOI: 10.3724/SP.J.1123.2020.05028 Abstract ( 148 )   HTML ( 41 )   PDF (3750KB) ( 68 )   Proteomics is an emerging field that has been shown to play a crucial role in unveiling the mechanisms underlying physiological and pathological processes, and liquid chromatography-mass spectrometry (LC-MS) is one of the most important methods employed in this field. However, in complex biological systems, such as eukaryotes, it is challenging to perform a comprehensive and unbiased proteome analysis due to the high complexity of biological samples and enormous differences in sample contents. For example, post-translational modifications (PTMs) in proteins are imperative for cell signaling, but post-translationally modified proteins account for about 1% of the total proteins in a single cell, making their identification extremely difficult. Therefore, chromatographic separation methods based on different principles are generally applied to reduce the complexity of biological samples and enrich trace proteins for their identification through mass spectrometry (MS). In this study, we developed a new proteomics method by combining size exclusion chromatography (SEC) and reversed-phase chromatography (RPLC), to separate and identify trace proteins in complex systems. SEC was used to separate and enrich kidney-specific proteins. After optimization of the method, it was found that 30 mmol/L of ammonium acetate could efficiently separate rat kidney proteins from the total protein fraction so that they could be eluted based on their relative molecular mass. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and LC-MS results showed that our SEC separation method not only refined the protein composition of the biological sample but also enhanced the relative contents of trace proteins through multiple injections. The collected protein fractions were further concentrated through ultrafiltration centrifugation followed by freeze-drying, which further improved the recovery of trace proteins by approximately 90% and largely decreased the time required with the use of freeze-drying alone. Thereafter, five protein fractions were separately digested using trypsin, and the resultant peptides were further analyzed by reverse phase chromatography-MS analysis. In the RPLC column, the peptides were isolated mainly based on their hydrophobicity. As a result, by combining SEC and RPLC, 23621 peptides and 1345 proteins were identified from the kidney, with an increase in numbers by 69% and 27%, respectively, when compared to those obtained using the common 2D strong cation exchange (SCX)-RPLC-MS method. However, no significant difference was observed in the pI and grand average of hydropathicity (GRAVY) values. Gene ontology (GO) analysis revealed an increase in the number of proteins in each cell component, especially the membrane. Furthermore, identification of a higher rate of identified peptides than proteins suggested that the protein coverage was also improved, thereby facilitating the detection of PTM proteins. Consequently, five common PTMs in biological processes, including methylation, acetylation, carbamylation, oxidation, and phosphorylation, were examined and compared between the two methods. As expected, the number of post-translationally modified peptides identified using SEC-RPLC-MS were 1.7-1.9 times more than those determined using the SCX-RPLC-MS method. Especially for the identification of phosphorylated peptides, we could achieve the level of the targeted enrichment strategy; however no significant difference was observed in the extents of phosphorylation among serine, threonine, and tyrosine. These results further indicate that upon combining SEC and RPLC, high efficiency could be achieved by decreasing the complexity of the protein sample, and the identification was unbiased. Finally, the phosphorylation of some kidney proteins, such as spectrin, L-lactate dehydrogenase, and ATPases, was found, which is critical for their functions. In summary, the SEC-RPLC-MS approach was developed for the identification of rat kidney proteins and is especially applicable for the identification of PTM proteins. Using this method, the identification efficiency for PTM peptides increased significantly. Therefore, this method has potential for better understanding the impact of PTM on kidney proteins and further elucidating the potential mechanisms underlying its physiological and pathological functions.

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Determination of nine N-nitrosamines in animal derived foods by QuEChERS-isotope dilution combined with gas chromatography-tandem mass spectrometry KONG Xiangyi, ZHUANG Lili, FANG Enhua, LIN Peng, ZHENG Zilong, ZHENG Xianghua, XU Dunming 2021, 39 (1):  96-103.  DOI: 10.3724/SP.J.1123.2020.06010 Abstract ( 204 )   HTML ( 56 )   PDF (1431KB) ( 180 )   In this study, a comprehensive analytical method based on gas chromatography-tandem mass spectrometry (GC-MS/MS) was developed for the determination of nine N-nitrosamines in animal derived foods. There are many kinds of N-nitrosamines in foods that are harmful to human health. However, the national standard GB 5009.26-2016 pertains only to the detection of N-dimethylnitrosamine; there are many drawbacks of this method, such as complicated sample preparation, low recovery rate, and poor reproducibility. Hence, it is of practical significance to establish a method for the simultaneous determination of a variety of N-nitrosamines. The optimal extraction conditions for the developed method were as follows: 10.0 g aliquots of the sample were placed in a 50 mL centrifuge tube, followed by the addition of 10 mL acetonitrile and 200 μL internal working standard solutions. After 30 min of freezing treatment, 4 g magnesium sulfate and 1 g sodium chloride were added for dehydration, and the tube was centrifuged at 9000 r/min for 5 min. After vortex centrifugation, 5 mL of the clear supernatant was purified using 150 mg polystyrene divinylbenzene (PLS-A). The purified extracts were dewatered using 1.6 g MgSO4 and 0.4 g NaCl, and then filtered through a 0.22 μm membrane filter unit prior to GC-MS/MS analysis. Temperature-programmed was applied at an initial temperature of 50 ℃. After 0.16 min, the temperature was raised to 220 ℃ at the rate of 900 ℃/min for 5 min. N-Nitrosamines were separated on an HP-Innowax column (30 m×0.25 mm×0.25 μm). Identification and quantification were achieved using an electron impact ion (EI) source in positive ion mode with multiple reaction monitoring (MRM). The internal standard method was used to quantify the N-nitrosamines. Under the optimal conditions, the correlation coefficients of the standard calibration curves were not less than 0.99 in the range of 0.1-50.0 μg/L. The limits of detection were 0.03-0.30 μg/kg (S/N=3), and limits of quantification were 0.15-1.00 μg/kg (S/N=10). At spiked levels of 0.5, 1.0, and 3.0 μg/kg, the average recoveries of N-nitrosamines in spiked samples ranged from 80.4% to 98.5%, with relative standard deviations between 2.41% and 12.50%. This method was used to determine animal derived food products, except N-itrosomethylethylamine and N-nitrosomorpholine, others were founded. The results showed that N-nitrosamines levels in salted aquatic products were generally higher than those of the other samples. The method established in this study is simple to operate, and it does not require any time-consuming distillation extraction. Furthermore, there is minimal consumption of samples and reagents; consequently, the experiment cost is reduced, and the method is environmentally friendly. This method has theoretical and practical significance for the control of N-nitrosamines residues in animal derived foods, establishment of detection standards, and corresponding management measures.