Hydrophilic Interaction Chromatography Mass Spectrometry Research Articles

Metabolomic Profiling Confirms the Key Role of Oxidative Stress in the Pathophysiology of Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA), a rare congenital bone marrow failure syndrome, characterized by severe anemia, erythroblastopenia, and congenital malformations is mostly associated with pathogenic variants in ribosomal protein (RP) genes, which cause their haploinsufficiency. The marked heterogeneity of the disease, related to different severity of hematological and non-hematological symptoms and diverse response to treatment, even in patients harboring the same RP mutation is not fully understood. Similarly, the mechanisms responsible for the predisposition of DBA patients to cancer await elucidation. We previously showed that the formation of reactive oxygen species (ROS) exceeds the anti-oxidant capacity of DBA erythrocytes leading to their higher susceptibility to destruction. Using DBA patients' samples and CRISPR/Cas9-created Rps19- and Rpl5-haploinsufficient MEL cellular models we also showed, that excessive oxidative stress, DNA damage, and activation of DNA damage response of erythroblasts is associated with an increased production of pro-/inflammatory cytokines and induction of apoptosis and senescence. In recent years, the use of metabolomic analysis appears as a promising tool that could contribute to a better understanding of the pathophysiology of the disease and discovery of novel biochemical markers with their potential use in diagnostics and prognostication. We therefore performed targeted metabolomic analysis of serum and erythrocyte samples from DBA patients (n=26) and healthy controls (n=10) using hydrophilic interaction chromatography-mass spectrometry (HILIC-MS) where 129 metabolites were detected. Significant changes in the metabolome of DBA patients were observed, with the key deregulated metabolites involved in the antioxidant defense and energy metabolism; the reduction of amine compounds (creatinine, guanidinoacetate) and hippurate in DBA patients compared to controls was also noted. In two DBA families analyzed, the altered erythrocyte metabolism and markers of oxidative stress distinguished the patients from their asymptomatic family members with the same RP mutation. The incubation of erythrocytes from symptomatic DBA patients with antioxidant N-acetylcysteine in vitro led to normalization of ROS levels in erythrocytes. Similarly, the treatment of Rpl5- and Rps19-haploinsufficienct MEL cells with a pro-inflammatory cytokine inhibitor pomalidomide, resulted in the amelioration of oxidative DNA damage (assessed by the presence of 8-oxoguanine, 8-oxoG) and reduction of p53 activation. In addition, targeted metabolomic analysis of cellular extracts showed clear separation of Rps19- and Rpl5-haploinsufficient MEL cells from control cells with significant changes affecting the level of many carbohydrates and acylcarnitines. Elevated levels of glutathione and pentose phosphate cycle intermediates in Rps19- and Rpl5-haploinsufficient MEL cells correspond with their need to cope with increased oxidative stress. Significantly increased levels of deoxynucleotides, mainly in Rpl5-haploinsufficient MEL cells, could be related to DNA damage response (DDR) process to promote repair of ROS-mediated damaged DNA. Nucleotide formation is an energy intensive process and therefore the observed decrease in acylcarnitines could be related to increased beta oxidation and increased formation of reduced coenzymes. This result from model MEL cells is consistent with 8-oxoG foci and activation of ATM (a DDR kinase) observed in erythroblasts of DBA patients expanded in vitro. Importantly, immunoreactivity for 8-oxoG and phospho-ATM S1981 was detected also in non-erythroid bone marrow cells of two DBA patients, who recently developed myelodysplastic neoplasia (MDS). Collectively, our data support the involvement of oxidative stress and pro-/inflammatory cytokines in the pathophysiology of DBA and suggest that the treatment aiming to reduce oxidative stress and/or suppress inflammation could alleviate the severity of the disease, though additional studies are needed. The potential link between oxidative damage, inflammatory cytokines, and cancer risk in DBA also deserves further research. Grant support: The Ministry of Education, Youth and Sports of the Czech Republic (8F20005), European Union - Next Generation EU, Program EXCELES (LX22NPO5102); Internal grant of Palacky University (IGA_LF_2023_002).

2176. Cardiolipin Synthases Play Redundant Roles And Are Required for Membrane Remodeling in Daptomycin Resistant Enterococcus faecalis

Abstract Background Daptomycin (DAP) is a lipopeptide antibiotic targeting anionic phospholipids (APLs) at the division septum. Resistance to DAP (DAP-R) in E. faecalis (Efs) has been associated with activation of the LiaFSR response and redistribution of APL microdomains through the effector protein LiaX. We also identified a novel transmembrane protein (designated LiaY) as essential for changes in APL microdomains which contain cardiolipin (CL). Efs encodes two CL synthase genes, cls1 and cls2. While changes in Cls1 are associated with DAP-R, their link to the LiaFSR system remains unclear. We aim to establish the roles of Cls in DAP-R to provide insights into membrane remodeling associated with DAP-R. Methods qRT-PCR was used to study cls expression. Membrane lipid content was analyzed using hydrophilic interaction chromatography-mass spectrometry. Mutants were characterized by DAP minimum inhibitory concentration (MIC) using E-test and localization of APL microdomains with 10-N-nonyl-acridine orange. The bacterial two hybrid system was used to evaluate protein interaction. Fluorescence microscopy evaluated protein co-localization. Results cls1 was upregulated in DAP-R Efs OG117ΔliaX. Deletion of either cls caused upregulation of the remaining cls gene. Development of DAP-R altered membrane lipid content, namely, an increase in CL and a shift towards species with longer fatty acid chains and higher degree of unsaturation. Upon deletion of both cls, CL content was eliminated, peripheral APL microdomains were abolished, and a decrease in the DAP MIC was observed. LiaY interacted with Cls1, but not Cls2 using the two-hybrid system. The interaction was confirmed with fluorescence microscopy showing co-localization of LiaY and Cls1 at the septum and away from the septum in DAP-S and DAP-R strains, respectively. LiaY and Cls1 were also individually co-localized to APL microdomains. Conclusion While Cls1 is predominantly associated with DAP-R, we show a functional redundancy between Cls1 and Cls2 in both membrane homeostasis and in DAP-R. Cls1 interacts with LiaY, which bridges the resistance mechanism with membrane adaptation, ultimately leading to altered localization of APL microdomains that divert DAP away from the division septum. Disclosures William R. Miller, M.D., Merck: Grant/Research Support|UpToDate: Honoraria

Comprehensive Screening for EPA/DHA-Structured Phospholipids in Aquatic Products by a Specific Precursor Ion Scanning-Based HILIC-MS/MS Method

Comprehensive screening for functional substances from natural resources is always a hot research topic. Eicosapentaenoic acid- (EPA) and docosahexaenoic acid (DHA)-structured phospholipids (PLEPA/DHA) have versatile cardiovascular benefits as well as superior bioavailability. Herein, the abundance of PLEPA/DHA in 16 aquatic products was specifically and selectively screened using a recently developed precursor ion scan-driven hydrophilic interaction chromatography-mass spectrometry (PreIS-HILIC/MS) method with the fatty acyl moieties of EPA (m/z 301.6) and DHA (m/z 327.6) locked. The aim focused on the characteristics and differences in the varieties and contents of EPA/DHA-structured phosphatidylcholine (PCEPA/DHA) and EPA/DHA-structured phosphatidylethanolamine (PEEPA/DHA) molecular species. A total of 80 PLEPA/DHA molecules were identified in these natural sources, including 47 PCEPA/DHA and 33 PEEPA/DHA. After analysis, PC 16:0/20:5 and PC 16:0/22:6 are present in all aquatic products and at high levels. Antarctic krill was found to be the best resource of PLEPA/DHA in total (2574.69 μg·g-1), followed by mackerel (2330.11 μg·g-1), salmon (2109.91 μg·g-1), and Farrer's scallop (1883.59 μg·g-1), while abalone contained the lowest level of PLEPA/DHA (310.44 μg·g-1). Besides, sea cucumber and sea bass contained the highest contents of EPA-structured and DHA-structured ether phospholipids, respectively, which could be highly recommended as dietary sources of special functional phospholipids. Finally, the multiple discrepancies between the 16 aquatic products were revealed by multivariate statistical analysis. These findings improve the awareness of the composition and content of PLEPA/DHA contained in aquatic products, providing a reference for their integrated development.

140. Enterococcus faecalis CL Synthases Have Redundant Roles and Play A Major Role in Phospholipid Redistribution Associated with Daptomycin Resistance

Abstract Background Daptomycin (DAP) is a lipopeptide antibiotic targeting membrane anionic phospholipids (APLs) at the division septum. DAP resistance (DAP-R) has been associated with activation of the LiaFSR system resulting in redistribution of APL microdomains (likely containing cardiolipin, CL) away from the septum. E. faecalis (Efs) possess two CL synthase genes, (cls1 and cls2) and changes in Cls1 are associated with DAP-R. However, the roles of each enzyme are unknown. Here, we characterize the roles of cls genes in DAP-R in the context of LiaFSR activation. Methods cls1 and/or cls2 were deleted from Efs OG117 and OG117ΔliaX (DAP-R strain with an activated LiaFSR response). qRT-PCR was used to study gene expression of cls1 and cls2 in the cls mutants. Membrane lipid content was analyzed using hydrophilic interaction chromatography-mass spectrometry. Mutants were characterized by DAP minimum inhibitory concentration (MIC) using E-test and localization of APL microdomains with 10-N-nonyl-acridine orange. Results cls1 and cls2 are upregulated in exponential phase of DAP-R Efs OG117ΔliaX relative to DAP-S Efs OG117, with only cls1 upregulated in stationary phase. Deletion of cls1 or cls2 resulted in upregulation of the other cls gene, independent of activation of LiaFSR. Lipidomics analysis confirmed that deletion of both cls resulted in complete absence of cell membrane CL content. When comparing CL profiles of Δcls1 relative to Δcls2 in both DAP-S and DAP-R, both strains produced similar levels and species of CL. However, development of DAP-R caused a change in membrane lipid content, namely, an increase in CL with no significant difference in phosphatidylglycerol compared to DAP-S strain. Evaluation of CL species in DAP-R shows a shift towards species containing longer fatty acid chains and higher saturation. Independent deletions of cls1 or cls2 did not revert the DAP phenotype. In contrast, deletion of both cls genes decreased the DAP MIC (2-3 fold) relative to the parent strain and restored septal localization of APL microdomains. DAP MIC was restored upon trans complementation of either cls1 or cls2 into the double deletion mutant. Conclusion Our results support a major role of Cls in changes in cell membrane architecture and DAP-R in enterococci, with overlapping roles for Cls1 and Cls2. Disclosures Cesar A. Arias, MD, PhD, Entasis Phramceuticals: Grant/Research Support|MeMed Diagnostics: Grant/Research Support|Merck: Grant/Research Support.

Simultaneous determination of nereistoxin insecticides in foods of animal origins by combining pH-dependent reversible partitioning with hydrophilic interaction chromatography-mass spectrometry

Although nereistoxin insecticides (NIs) are banned for animal husbandry operations, they are still used because of their high insecticidal activities. Therefore, a reliable residue analysis method for the simultaneous detection of cartap, bensultap, thiocyclam, and nereistoxin in foods of animal origins, including beef, pork, chicken, milk, and eggs, was developed using hydrophilic interaction liquid chromatography–mass spectrometry (HILIC–LC–MS/MS). The NIs were extracted with an acidic cysteine and formate buffer solution and hydrolyzed to nereistoxin. The molarity and pH of the buffer were optimized at 20 mM and 3, respectively, to keep the pH of the extracts at 4–5. pH-dependent acid–base partitioning coupled with salting-out-assisted liquid–liquid extraction using acetonitrile was performed for purification and for the direct introduction of the extracts to LC. The optimal pH values were 5 and 9 for the acid–base partitioning. Nereistoxin quantitation was achieved with consistent column retention (RSD < 0.6%) and a high degree of separation (N > 106). The matrix-dependent method limit of quantitation was 2 μg nereistoxin/kg, and the calibration curve showed good linearity (R2 > 0.998). The recovery efficiencies were in the range of 89.2–109.9% with relative standard deviations less than 10%, and matrix effects did not exceed ± 10%, which satisfied the criteria outlined in the European SANTE/12682/2019 guidelines.

Screening of Phospholipids in Plasma of Large-Artery Atherosclerotic and Cardioembolic Stroke Patients With Hydrophilic Interaction Chromatography-Mass Spectrometry.

Ischemic stroke (IS) is a deadly and debilitating disease with a high incidence and recurrence rate in elderly people worldwide. Large-artery atherosclerotic (LAA) and cardioembolic (CE) stroke are two leading subtypes and require different management. As a complementary biochemistry method for current diagnostic techniques, a sensitive and accurate phospholipid (PL) targeted lipidomic method was developed in this study. Plasma PLs were selectively extracted with titanium dioxide/fibrous silica nanosphere material, then characterized and quantified with hydrophilic interaction chromatography-mass spectrometry. A total of 31 molecular species of PLs were determined and ten biomarkers including seven molecular species of sphingomyelins (SM d18:1/18:1, d18:1/18:0, d18:1/24:1, d18:1/16:1, d18:1/22:1, d18:1/24:2, and d18:1/16:0) and three molecular species of phosphatidylcholines (16:0/18:1, 16:0/18:2 and 16:0/22:6) showed significant differences in LAA, CE, and healthy control (HC) groups. The independent diagnostic capabilities of these PL biomarkers were successfully evaluated and validated with receiver operating characteristic curves. Additionally, the oleic acid-enriched SMs, which can result in atherogenic lipoprotein aggregation, were proved to be positively related to IS and may perform as the potential risk factors in the future. Meanwhile, valuable suggestions for dietary interventions as an essential source of endogenous PLs could be obtained from this study.

1275. Dynamics of Enterococcus faecalis Cardiolipin Synthase Gene Expression Reveal Compensatory Roles in Daptomycin Resistance

BackgroundDaptomycin (DAP) is a lipopeptide antibiotic targeting membrane anionic phospholipids (APLs) at the division septum, and resistance (DAP-R) has been associated with activation of the E. faecalis (Efs) LiaFSR response and redistribution of APL microdomains (predicted to contain cardiolipin) away from the septum. Efs encodes two putative cardiolipin synthase genes, cls1 and cls2. While changes in Cls1 are associated with DAP-R, the exact roles of each enzyme in resistance are unknown. This work aims to establish the contributions for both enzymes in the development of DAP-R.Methods cls1 and cls2 were deleted individually and in tandem from Efs OG117∆liaX (a DAP-R strain with an activated LiaFSR response). Mutants were characterized by DAP minimum inhibitory concentration (MIC) using E-test and localization of APL microdomains with 10-N-nonyl-acridine orange staining. Quantitative PCR (qRT-PCR) was used to study gene expression profiles of cls1 and cls2 in Efs OG117∆liaX relative to Efs OG117. Membrane lipid content was analyzed using hydrophilic interaction chromatography-mass spectrometry (HILIC-MS). Results cls1 was highly upregulated in stationary phase concurrent with a decrease in cls2 expression. However, independent deletion of cls1 or cls2 in the DAP-R background resulted in no significant phenotypic changes from the parent strain. Interestingly, qRT-PCR showed that cls2 expression was upregulated upon deletion of cls1 (and vice-versa), suggesting a compensatory role for one enzyme upon deletion of the other (Fig 1). When comparing membrane lipid content between Efs OG117∆liaX∆cls1 and Efs OG117∆liaX∆cls2, there were no significant differences in both the overall amount or species of cardiolipin generated, further supporting a potential redundancy between the cardiolipin synthases (Fig 2). Ultimately, double deletion of both cls genes lowered the DAP MIC relative to the parent strain and restored septal localization of APL microdomains. ConclusionOverall, Cls1 has a predominant role in the development of DAP-R in E. faecalis. However, here, we describe a novel compensatory role for Cls2 under conditions in which there is no functional Cls1 to maintain the DAP-R phenotype. Disclosures Truc T. Tran, PharmD, Merck (Grant/Research Support) Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support)

Lipidomics Screening of Polyunsaturated Phospholipid Molecular Species in Crab (Portunus trituberculatus) Muscular Tissue: A Nontarget Approach by HILIC‐MS

AbstractThe phospholipid molecular species (PMS) in the muscular tissue of the swimming crab (Portunus trituberculatus) are extracted and quantified using aminopropyl coated silica‐based solid‐phase extraction (SPE) and hydrophilic interaction chromatography‐mass spectrometry (HILIC‐MS), respectively. Once the lipidomics profile is acquired, the contents of PMS are semiquantitatively determined using linear regression models. It is found that in swimming crab, phosphatidylcholine (PC, 14.30 mg g−1) is the most abundant phospholipid class, followed by phosphatidylethanolamine (PE, 8.20 mg g−1), phosphatidylinositol (PI, 1.60 mg g−1), and phosphatidylserine (PS, 9.83 mg g−1). The nutritional eicosapentaenoic acid and/or docosahexaenoic acid structured phospholipids exist in swimming crabs, such as PC 18:0/20:5, PE 18:1/20:5 and 16:0/22:6, PI 18:0/20:5. Besides, plasmalogens are detected, such as PC o‐16:0/22:6 and PE o‐18:0/20:5. Finally, this method is validated to be efficient and accurate in terms of linearity (correlation coefficients, R2 0.9976 to 0.9993), sensitivity (limit of detection, LOD, 0.22–0.33 µg mL−1), precision (relative standard deviation, RSDintra‐day ≤ 4.11%), and recovery (78% to 85%, RSD ≤ 5.79%). In conclusion, swimming crab is rich in health‐beneficial phospholipids, such as eicosapentaenoic acid and/or docosahexaenoic acid structured phospholipids and plasmalogens, and the muscular tissue is proved to be of high nutritional value.Practical Applications: The study introduces a potential method for the lipidomics screening of polyunsaturated phospholipid molecular species in food.

Comparison of Different Labeling Techniques for the LC-MS Profiling of Human Milk Oligosaccharides.

Human milk oligosaccharides (HMOs) exhibit various biological activities for infants, such as serving as prebiotics, blocking pathogens, and aiding in brain development. HMOs are a complex mixture of hetero-oligosaccharides that are generally highly branched, containing multiple structural isomers and no intrinsic chromophores, presenting a challenge to both their resolution and quantitative detection. While liquid chromatography-mass spectrometry (LC-MS) has become the primary strategy for analysis of various compounds, the very polar and chromophore-free properties of native glycans hinder their separation in LC and ionization in MS. Various labeling approaches have been developed to achieve separation of glycans with higher resolution and greater sensitivity of detection. Here, we compared five commonly used labeling techniques [by 2-aminobenzamide, 2-aminopyridine, 2-aminobenzoic acid (2-AA), 2,6-diaminopyridine, and 1-phenyl-3-methyl-5-pyrazolone] for analyzing HMOs specifically under hydrophilic-interaction chromatography-mass spectrometry (HILIC-MS) conditions. The 2-AA labeling showed the most consistent deprotonated molecular ions, the enhanced sensitivity with the least structural selectivity, and the sequencing-informative tandem MS fragmentation spectra for the widest range of HMOs; therefore, this labeling technique was selected for further optimization under the porous graphitized carbon chromatography-mass spectrometry (PGC-MS) conditions. The combination strategy of 2-AA labeling and PGC-MS techniques provided online decontamination (removal of excess 2-AA, salts, and lactose) and resolute detection of many HMOs, enabling us to characterize the profiles of complicated HMO mixtures comprehensively in a simple protocol.

Fast and Specific Screening of EPA/DHA-Enriched Phospholipids in Fish Oil Extracted from Different Species by HILIC-MS.

Eicosapentaenoic acid- and docosahexaenoic acid-enriched phospholipids (PLEPA/DHA) have versatile health-beneficial functions and can be well absorbed in the intestine. Herein, a precursor ion scan-driven hydrophilic interaction chromatography mass spectrometry (PreIS-HILIC-MS) method with the fatty acyl moieties of m/z 301.6 and 327.6 locked was established to specifically and selectively screen PLEPA/DHA in different fish oil samples, including saury, grass carp, hairtail, and yellow croaker. Taking saury oil as an example, a total of 24 PLEPA/DHA were successfully identified and quantified, including 20 PCEPA/DHA and 4 PEEPA/DHA. Finally, this method was validated in terms of sensitivity (limit of detection ≤ 4.15 μg·mL-1), linearity (≥0.9979), precision (RSDintraday ≤ 4.65%), and recovery (≥78.6%). The performance of the PreIS-HILIC-MS method was also compared with that of the traditional full-scan mode, and the former demonstrated its unique superiority in targeted screening of PLEPA/DHA in fish oils.

Compositional study of plasmalogens in clam (Corbicula fluminea) by TiO2/KCC-1 extraction, enzymatic purification, and lipidomics analysis

In this study, the plasmalogens in clam (Corbicula fluminea) were extracted by titanium dioxide (TiO2) fibrous silica nanosphere (KCC-1) extraction and phospholipase A1 hydrolysis. The molecular composition of plasmalogens was lipidomically analyzed using hydrophilic interaction chromatography mass spectrometry (HILIC-MS). The results showed that there was a total of 34 plasmalogen molecular species structurally elucidated. The quantitative results of linear regression models indicated that plasmenylcholine (14.24 μg mg–1) was the most abundant class, followed by plasmenylethanolamine (11.25 μg mg–1), and plasmenylserine (9.78 μg mg–1). Multivariate data analysis was applied to reduce data dimensions and circos map was used to provide an integrated and holistic view of correlation coefficients between the samples and plasmalogen molecular species. This method was validated to be accurate and sensitive in extraction and analysis of plasmalogens in clams.

Lipidomics phenotyping of clam (Corbicula fluminea) through graphene/fibrous silica nanohybrids based solid-phase extraction and HILIC-MS analysis

Polyunsaturated phospholipids are abundant in clam (Corbicula fluminea) but difficult to be fully extracted. Herein, graphene/fibrous silica (G/KCC-1) nanohybrids were synthesized, characterized, and applied for solid-phase extraction (SPE) of phospholipids in clam. The effectiveness of G/KCC-1 SPE was verified by hydrophilic interaction chromatography mass spectrometry (HILIC-MS) based lipidomics and statistical analysis. The ions of PE 16:0/18:1 (m/z 716.4), PC 16:0/20:5 (m/z 824.6) and etc. were regarded as the main difference among the crude lipids, acetone washed extract, and eluate of G/KCC-1 SPE. Finally, this method was validated in terms of linearity (R2 0.9965 to 0.9981), sensitivity (LOD 0.19–0.51 μg·mL−1 and LOQ 0.48 – 1.47 μg·mL−1), and precision (RSDintra-day ≤ 7.16% and RSDinter-day ≤ 7.30%). In conclusion, the G/KCC-1 SPE and HILIC-MS method was shown to be accurate and efficient in selective extracting and phenotyping phospholipids in C. fluminea.

Benchmarking Non-Targeted Metabolomics Using Yeast-Derived Libraries.

Non-targeted analysis by high-resolution mass spectrometry (HRMS) is an essential discovery tool in metabolomics. To date, standardization and validation remain a challenge. Community-wide accepted cost-effective benchmark materials are lacking. In this work, we propose yeast (Pichia pastoris) extracts derived from fully controlled fermentations for this purpose. We established an open-source metabolite library of >200 identified metabolites based on compound identification by accurate mass, matching retention times, and MS/MS, as well as a comprehensive literature search. The library includes metabolites from the classes of (1) organic acids and derivatives (2) nucleosides, nucleotides, and analogs, (3) lipids and lipid-like molecules, (4) organic oxygen compounds, (5) organoheterocyclic compounds, (6) organic nitrogen compounds, and (7) benzoids at expected concentrations ranges of sub-nM to µM. As yeast is a eukaryotic organism, key regulatory elements are highly conserved between yeast and all annotated metabolites were also reported in the human metabolome database (HMDB). Orthogonal state-of-the-art reversed-phase (RP-) and hydrophilic interaction chromatography mass spectrometry (HILIC-MS) non-targeted analysis and authentic standards revealed that 104 out of the 206 confirmed metabolites were reproducibly recovered and stable over the course of three years when stored at −80 °C. Overall, 67 out of these 104 metabolites were identified with comparably stable areas over all three yeast fermentation and are the ideal starting point for benchmarking experiments. The provided yeast benchmark material enabled not only to test for the chemical space and coverage upon method implementation and developments but also allowed in-house routines for instrumental performance tests. Transferring the quality control strategy of proteomics workflows based on the number of protein identification in HeLa extracts, metabolite IDs in the yeast benchmarking material can be used as metabolomics quality control. Finally, the benchmark material opens new avenues for batch-to-batch corrections in large-scale non-targeted metabolomics studies.

Real-Time In Situ Screening of Omega-7 Phospholipids in Marine Biological Resources Using an iKnife-Rapid-Evaporative-Ionization-Mass-Spectrometry-Based Lipidomics Phenotype.

Omega-7 (n-7) phospholipids were bioactive substances in marine animals. In this study, a fast lipidomics phenotyping approach for real-time in situ screening of n-7 phospholipids in five kinds of economic seafood, salmon, prawn, bluefin tuna, hairtail, and butterfish, was established using iKnife rapid evaporative ionization mass spectrometry (REIMS). The n-7 phospholipids were structurally characterized and quantitatively analyzed, and the profiles were statistically analyzed by multivariate recognition analysis. It indicated that the difference of n-7 phospholipids in seafood samples was significant (p < 0.05), with R2(cum) and Q2(cum) values of >0.9. The proportion of n-7 phospholipids in salmon was the highest (20.43%), followed by bluefin tuna, prawn, hairtail, and butterfish. The ions of m/z 742.54 (PC 16:1-18:1), 768.55 (PC 16:1-20:2), 697.48 (PE 16:1-18:1), and 699.48 (PE 16:1-18:0) were the main n-7 phospholipids. The effectiveness of iKnife REIMS was further verified by hydrophilic interaction chromatography mass spectrometry and gas chromatography. The results demonstrated that proposed iKnife REIMS was an excellent technique for front-line screening of n-7 phospholipids in a large variety of marine biological resources.

Glycine additive facilitates site-specific glycosylation profiling of biopharmaceuticals by ion-pairing hydrophilic interaction chromatography mass spectrometry.

Many biotherapeutics such as monoclonal antibodies (mAb) and Fc-domain fusion proteins contain heterogeneous glycan contents at one or multiple glycosylation site(s). Site-specific glycan profile characterization is critical for monitoring the quality of these molecules during different stages of drug development. Hydrophilic interaction chromatography (HILIC) as an orthogonal separation method to reversed-phase liquid chromatography (RPLC) can achieve better glycopeptide identification due to the effective separation between individual glycoforms as well as the separation of glycopeptides from high-abundance non-glycosylated peptides, which can be further improved by modifying the mobile phases with ion-pairing agents (IP-HILIC). However, an online IP-HILIC coupled to mass spectrometry (MS) detection may suffer from the suppression of mass spectrometry signal during electrospray ionization due to the trifluoroacetic acid (TFA), commonly used as an ion-pairing agent. Here, we reported an optimized experimental condition for IP-HILIC-MS where glycine is added in the TFA-containing mobile phases to enhance the MS detection sensitivity for glycopeptides up to ~ 50-fold by eliminating the ion-suppression effect of an ion-pairing agent while still retaining excellent separation capacity. We demonstrated that with enhanced detection sensitivity, IP-HILIC-MS can confidently identify an increased number of site-specific N-linked glycans for IgG1, and IgG4 mAbs as well as an Fc-domain fusion protein (containing five N-glycosylation sites) through MS/MS-based search in the data-dependent acquisition mode, meanwhile, achieve comparable quantitative results compared with the traditional methods. We also demonstrated that IP-HILIC-MS can be used to identify low-level O-glycosylation and non-consensus N-glycosylation on mAbs without any enrichment prior to LC-MS analysis. Graphical abstract.

Monodisperse microsphere-based immobilized metal affinity chromatography approach for preparing Antarctic krill phospholipids followed by HILIC-MS analysis

Phospholipids enriched krill is a functional food beneficial in cardiovascular diseases. Herein, monodisperse microsphere-based immobilized metal affinity chromatographic material (MM-IMAC) was synthesized with Ti4+ incorporated to enrich phospholipids from krill by coordination with phosphate group. The extract was profiled by hydrophilic interaction chromatography-mass spectrometry (HILIC-MS) with 154 phospholipid molecular species detected. The parameters were loading solvent n-hexane/isopropanol (2:8, v/v), flow rate 0.8 mL·min−1, and eluting volume 1 mL. Besides, eicosapentaenoic and docosahexaenoic acids structured phospholipids were located, such as phosphatidylcholine (PC) 20:5/22:6, phosphatidylinositol (PI) 18:0/20:5, etc. Finally, this method was validated in linearity (R2 ≥ 0.9953), sensitivity (LOD ≤ 0.53 μg·mL−1 and LOQ ≤ 1.66 μg·mL−1), precision (RSDintraday ≤ 4.86% and RSDinterday ≤ 6.25%), and recovery (58–83%). It indicated that the MM-Ti4+-IMAC-HILIC-MS was reliable and efficient in specific study of phospholipids in food matrix.

Trace-level quantification of N-nitrosopiperazine in treated wastewater using supported liquid extraction and hydrophilic interaction chromatography mass spectrometry

Regenerable amine-based solvents used for post-combustion CO2 capture, primarily monoethanolamine and piperazine, are known to undergo degradation and secondary reactions over time forming, amongst other species, N-nitrosamines. These carcinogenic species can eventually make their way from treated wastewater into environmental waters. The United States Environmental Protection Agency (US EPA) recommends that the concentration of N-nitrosamines in surface water not exceed 1.24 μg/L. We have developed a straightforward method to quantify N-nitrosopiperazine in treated wastewater by hydrophilic interaction liquid chromatography – mass spectrometry (HILIC–MS) after sample preparation by supported liquid extraction (SLE). To achieve the best extraction recovery and method limits of quantification (MLOQ), standards were prepared in a high-salt synthetic matrix to mimic the treated wastewater effluent. To further improve the MLOQ, the drying steps after extraction were optimized. HILIC separation of the highly polar analytes was achieved using an ethylene-bridged hybrid amide stationary phase. Detection was achieved using a triple quadrupole mass spectrometer operated in positive electrospray ionisation and multiple reaction monitoring mode, providing a final MLOQ of 0.25 μg/L for N-nitrosopiperazine. Validation of the method was carried out to ensure good confidence in the data obtained for a treated wastewater sample from a post-combustion CO2 capture facility. In addition, N-nitrosopiperazine was quantified with the developed SLE-HILIC–MS method in eight degraded carbon capture samples that had not yet undergone wastewater treatment.

Quantitative and comparative study of plasmalogen molecular species in six edible shellfishes by hydrophilic interaction chromatography mass spectrometry

Shellfishes contain plasmalogens correlating to the functions of brain, heart, etc. Herein, a mild acid hydrolysis and hydrophilic interaction chromatography (HILIC) tandem mass spectrometry method was developed for analyzing plasmalogens in six shellfish species. A total of 19 plasmalogen molecular species were successfully identified, including nine phosphatidylcholine plasmalogen (plasPC), seven phosphatidylethanolamine plasmalogen (plasPE), and three phosphatidylserine plasmalogen (plasPS). The quantitative results indicated that mussel (32 μg·mg−1) possessed the highest content of plasmalogens, followed by oyster (21 μg·mg−1) and razor clam (15 μg·mg−1). The statistic models showed that the plasPE P-18:0/20:5 (m/z 748), plasPE P-16:0/22:2 & P-18:0/20:2 (m/z 754) and plasPS were the most contributing difference between shellfishes. The results indicated that this method was sensitive and precise to determine plasmalogens in shellfish, and mussel was demonstrated to be a good choice for the large-scale preparation of plasmalogens.

Triazole Hydrophilic Interaction Chromatography Mass Spectrometry–Based Method for Studying the Lipidomic Composition of Largemouth Bass (Micropterus salmoides) with Different Feeds

A triazole hydrophilic interaction chromatography mass spectrometry (HILIC-MS) method was developed for lipidomics studying the largemouth bass (Micropterus salmoides). The effect of feeds, especially the frozen trash fish and artificially formulated feed, on the phospholipid composition was evaluated. The results showed that the phospholipids could be well separated in the order of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol and phosphatidylserine. Under negative-ion mode, all the phospholipids were ionized by deprotonating as [M-H]− or adducting as [M + HCOO]−. The mass spectrum for each phospholipid class was inspected and a total of 34 phospholipid molecular species (PMS) was identified. Each PMS was extracted by its ion chromatography and quantified using a linear regression model. The calibration curves were linear ranging from 2.00 to 200 μg mL−1 (R2 0.9988–0.9991) with the limit of detection (LOD) and limit of quantitation (LOQ) of 0.15–0.52 μg mL−1 and 0.80–1.80 μg mL−1, respectively. The results indicated that the HILIC-MS method was efficient and reliable in lipidomics analyzing largemouth bass. After orthogonal partial least squares discriminant analysis (OPLS-DA), the major contributors to the difference of largemouth bass samples with different feeds were revealed as PE 760.8, PC 842.8, etc. and validated by permutation test with the intercepts of R2 = (0.0, 0.876) and Q2 = (0.0, − 1.21).

Untargeted HILIC-MS-Based Metabolomics Approach to Evaluate Coffee Roasting Process: Contributing to an Integrated Metabolomics Multiplatform.

An untargeted metabolomics strategy using hydrophilic interaction chromatography-mass spectrometry (HILIC-MS) was developed in this work enabling the study of the coffee roasting process. Green coffee beans and coffee beans submitted to three different roasting degrees (light, medium, and strong) were analyzed. Chromatographic separation was carried out using water containing 10 mM ammonium formate with 0.2 % formic acid (mobile phase A) and acetonitrile containing 10 mM ammonium formate with 0.2 % formic acid (mobile phase B). A total of 93 molecular features were considered from which 31 were chosen as the most statistically significant using variable in the projection values. 13 metabolites were tentatively identified as potential biomarkers of the coffee roasting process using this metabolomic platform. Results obtained in this work were complementary to those achieved using orthogonal techniques such as reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) and capillary electrophoresis-mass spectrometry (CE-MS) since only one metabolite was found to be common between HILIC-MS and RPLC-MS platforms (caffeoylshikimic acid isomer) and other between HILIC-MS and CE-MS platforms (choline). On the basis of these results, an untargeted metabolomics multiplatform is proposed in this work based on the integration of the three orthogonal techniques as a powerful tool to expand the coverage of the roasted coffee metabolome.