Sample Digestion Research Articles

Acute physiological effects on macromolecule digestion following oral ingestion of the enzyme blend Elevase® in individuals that had undergone an ileostomy, but were otherwise healthy-a randomized, double blinded, placebo-controlled exploratory study.

Digestive enzymes can selectively degrade proteins, carbohydrates and lipids; and their supplementation alongside food may accelerate the breakdown of complex food matrices, facilitate greater nutrient absorption, decrease food sensitivities and aid in the management of certain disease states. Several intrinsic and extrinsic factors govern food digestion and for every individual this phenomenon is unique. This study was conducted as a randomized, crossover, placebo-controlled design where each participant served as their own control. This post-hoc analysis investigated the impact of a dietary enzyme supplementation blend known as Elevase® on dietary macromolecule digestion in samples from otherwise healthy participants that had previously undergone a small bowel resection, resulting in an ileostomy (NCT04489810). This is the first time this study-paradigm has been used for the assessment of in vivo dietary breakdown following enzyme supplementation. Arguably, this technique offers superior data when compared to that generated in artificial gut digestion models, preclinical animal models, or indeed conventional clinical studies using stool analyses, as it allows real-time access to samples in situ in the small intestine where the majority of nutritional absorption takes place. It was demonstrated that after 4 h, Elevase® significantly increased monosaccharide levels (predominantly glucose and fructose) in the ileostomy samples taken from the same individuals on the same diet on a different day. In addition, the bile salt taurohyodeoxycholic acid was also increased, suggesting a physiological host response to the macromolecule digestion induced by the enzymatic blend. Overall, these findings suggest Elevase® could accelerate food digestion and potentially increase nutrient availability from the diet.

Possibilities of decontaminating organic waste from swine-farming complexes using anaerobic digestion

AbstractAnaerobic digestion with simultaneous production of biogas is one of the most common methods of recycling, processing and decontamination of organic waste to produce alternative fuels. A commonly used indicator of the sanitary safety of the substrate is eggs of Ascaris suum nematodes of pigs, which are characterised by extremely high resistance to environmental factors. The aim is to investigate the effect of the mesophilic mode of anaerobic digestion on the activity of pathogens, particularly Ascaris suum eggs. The eggs of the studied nematode species were placed in a biogas installation at a temperature of 37 °C. The digestate samples with eggs were then taken every 4 days for 28 days, followed by larvae culture to determine the viability of the eggs. The results of our research have shown that the mesophilic regime of anaerobic digestion is an effective method of controlling parasites, but at the same time, it needs to be improved, since only 7.6% of A. suum eggs remained alive after a 1-day stay, about 50% after the week stay and about 9% in the third week stay in the biogas reactor. Thus, further optimisation of anaerobic fermentation in the mesophilic mode can be aimed at improving the suppression of pathogenic activity.

Nutritional composition, protein digestibility and consumer acceptability of sorghum‐based mahewu enriched with Moringa oleifera leaf powder

SummaryIn this study, the effect of different levels (1%–5%) of whole and defatted Moringa oleifera leaf powder (MOLP) on the nutritional composition, physicochemical properties, protein digestibility and consumer acceptability of sorghum mahewu was investigated. The MOLP was boiled and added to the fermented sorghum mahewu. The results demonstrated that sorghum mahewu enriched with whole and defatted MOLP had higher amounts (P < 0.05) of protein, ash and fibre content compared to the control sample. Moreover, minerals, amino acid contents and protein digestibility of the enriched sorghum mahewu samples were significantly improved compared to the control sample. The pH decreased with an increasing proportion of MOLP ranging from 4.36 to 4.09 (mahewu enriched with whole MOLP) and from 4.36 to 4.17 (mahewu enriched with defatted MOLP). Sorghum mahewu samples enriched with whole and defatted MOLP had higher contents of total titratable acid compared to the control sample, while the total soluble solids and viscosity decreased. In terms of colour profile, the b*, and chroma values of sorghum mahewu increased with an increasing percentage of whole and defatted MOLP while L* and a* values decreased. Regarding overall liking, sorghum mahewu enriched with 1% whole and defatted MOLP did not differ (P > 0.05) from the control sample. Therefore, sorghum mahewu must be enriched with low percentages of MOLP to improve its acceptance among consumers. These findings suggest that incorporating medicinal plants such as boiled MOLP to basic foods produced from sorghum flour could improve their nutritional profile and protein digestibility.

Assessment of seasonal variations of some heavy metals in water samples collected from Gwaigwaye, Maska and Zobe dams

Water pollution can Damage Aquatic Ecosystems, leading to Death of Fish, plants and other Aquatic organism, which in turn can Disrupt the Entire Food chains. This study was conducted to Determine the Concentrations of Some Heavy metals in water samples obtained from Gwaigwaye, Maska and Zobe Dams in Dry and wet Seasons. The Dams provides water for Drinking, Fishing, Farming and other Agricultural Activities to the Neighbouring Communities. In this study water samples collected from Gwaigwaye, Maska and Zobe Dams in Dry and wet Seasons were Analyzed for Some Heavy metals namely, Cadmium, Cobalt, Copper, Nickel, lead and zinc. After Samples Digestion, microwave plasma atomic emission spectroscopy(MPAES) was used for the Determination of the Metal concentrations and their Levels were compared with permissible limits set by local and international standard regulatory Authorities. From the result of the Analysis, the Concentrations of the Heavy metals in Dry season in mg/L ranged as follows. 0.00±0.00 to 0.03±0.00 mg/L for Cd, 0.02±0.00 to 0.084±0.002 mg/L for Co, 0.057±0.003to 0.186±0.003 mg/L for Cu, 0.03±0.00 to 0.19±0.005 mg/L for Ni, 0.00±0.00 to 0.01±0.00 mg/L for Pb and 0.06±0.02-0.193±0.003 mg/L for Zinc. Similarly, the Concentrations of metals in wet season in(mg/L) ranged as follows, 0.00±0.00 to 0.027±0.002 for Cd, 0.02±0.002 to 0.071±0.002 for Co, 0.051±0.003 to 0.103±0.014 for Cu, 0.029±0.003 to 0.12±0.013 for Ni, 0.00±0.00 to 0.00±0.00 for pb and 0.057±0.002 to 0.160±0.003 for Zinc. The Concentrations level of Cd, Co and Ni in water samples from Maska, Gwaigwaye and Zobe Dams in dry season were above the permissible set by WHO/SON. However, Cu, Pb and Zn were within the permissible limit of WHO and SON in all the Dams in dry and wet seasons. The Result of Statistical Analysis indicated no Significant Difference between the Metals in all the Dams in both Dry and Wet seasons as p values were greater than 0.05( p >0.05).

Determination of the levels of selected essential and toxic metals in soil collected from the flower farm in Roshara Roses PLC (Bishoftu) of Ethiopia and assessment of associated human health risks

The overuse of pesticides and fertilizers to grow high-quality roses unintentionally introduces heavy metals into the soil which is detrimental to the human health. Thus, the aim of the current study is to examine the concentrations of the essential (Ca, Mg, Zn, Fe, Cu, Fe, Mn and Ni) and toxic metals (Cd and Pb) in the soil samples from the flower farm using Flame atomic absorption spectrometry (FAAS) and to assess the risk that heavy metals pose to human health. To obtain an optimum condition for the wet digestion of soil samples, the volume ratio of reagents, reflux time and temperature were optimized. The concentrations of Ca, Fe, Mn, Mg, Zn, Cr, Ni, Cu were found to be 3468, 4926, 450, 255, 74, 70,48.5 and 25 and 2150, 4862,472, 241, 71, 58, 37 and 12.5 mg/kg for the soil samples collected from the flower farm and road side, respectively. Cadmium and lead were not detected in the studied soil samples. Statistical analysis of variance (ANOVA) showed the concentrations of Ca and Cu in the two soil samples are significantly different. Correlation analysis showed there was strong positive association for Fe with Zn, Cr and Ni, Mg with Ca, Cu, Ni and Cr, Zn with Cr and Cu and Ni with (Cu, Zn and Cr). Values of hazard quotient (HQ) and hazard index (HI) for the heavy metals under the study in the all exposure pathways are below 1.0 indicating that none of them could be harmful to human health.

Optimization of vortex-assisted supramolecular solvent-based liquid liquid microextraction for the determination of mercury in real water and food samples

A novel method was developed for sample preparation for spectrophotometric determination of Hg(II) in water and food samples. The method was based on vortex-assisted supramolecular solvent-assisted liquid-liquid microextraction (VA-SUPRASs-LLME). Analytical parameters such as pH, chelating agent, solvent type and volume, vortex time and salting out effect were optimized. Surface and normal probability plots were drawn for the variables using the optimization data. Microwave-assisted digestion of samples was performed before the extraction procedure. L-cysteine was found to be more effective as a ligand for Hg(II). Five different SUPRASs were prepared and used for the extraction of Hg(II). A 1-decylamine/thymol/water at a 1:2:1 molar ratio assisted by a salting effect was found most effective for optimal extraction. Limits of detection and limit of quantification were found 0.6 μg L−1 and 2.0 μg L−1 with a very good linearity range of 2–350 μg L−1. Intra-day and inter-day precisions were in the range of 1.8–4.0 % with a preconcentration factor 150. The accuracy of the method estimated by analysis of certified reference materials was 96–98.5 %. Finally, the new method was used for the determination of Hg(II) in real water, food samples, and certified reference materials (NIST, IAEQ/W-4(simulated freshwater), and DORM-4; fish protein).

Robust assessment of sample preparation protocols for proteomics of cells and tissues

In proteomic studies, the reliability and reproducibility of results hinge on well-executed protein extraction and digestion protocols. Here, we systematically compared three established digestion methods for macrophages, namely filter-assisted sample preparation (FASP), in-solution, and in-gel digestion protocols. We also compared lyophilization and manual lysis for liver tissue protein extraction, each of them tested using either sodium deoxycholate (SDC)- or RIPA-based lysis buffer. For the macrophage cell line, FASP using passivated filter units outperformed the other tested methods regarding the number of identified peptides and proteins. However, a careful standardization has shown that all three methods can yield robust results across a wide range of starting material (even starting with 1 μg of proteins). Importantly, inter and intra-day coefficients of variance (CVs) were determined for all sample preparation protocols. Thus, the median inter-day CVs for in-solution, in-gel and FASP protocols were respectively 10, 8 and 9%, very similar to the median CVs obtained for the intra-day analysis (9, 8 and 8%, respectively). Moreover, FASP digestion presented 80% of proteins with a CV lower than 25%, followed closely by in-gel digestion (78%) and in-solution sample preparation (72%) protocols. For tissue proteomics, both manual lysis and lyophilization presented similar proteome coverage and reproducibility, but the efficiency of protein extraction depended on the lysis buffer used, with RIPA buffer showing better results. In conclusion, although each sample preparation method has its own particularity, they are all suited for successful proteomic experiments if a careful standardization of the sample preparation workflow is carried out.

Application of ICP-OES for determination of mercury species in environmental samples

Direct determination of mercury species in real environmental samples is challenging due to their trace concentrations and low ionisation energy. In this investigation, a microwave-assisted and aqua regia digestion method is proposed for the analysis of trace inorganic mercury. Measurements were performed using inductively coupled plasma optical emission spectroscopy (ICP-OES) method in soil, digestate and waste samples. The detection limits of the proposed method were found to be 0.03 and 0.115 mg/kg for mercury at 184.9 and 194.2 nm, respectively. The accuracy of the method with respect to the systematic and constant errors was confirmed by the analysis of certified reference material (CRM) (> 95 % recovery with < 15 % relative standard deviation RSD). The method was successfully applied for the determination of mercury in solid wastes listed in the Decree on Burdening of Soil with Waste Spreading and the Decree on the Treatment of Biodegradable Waste and the Use of Compost or Digestate in the Republic of Slovenia (Official Gazzette of the Republic of Slovenia, No. 34/08 and 61/11 and No 99/13, 56/15 and 56/18).

Identifikasi Mikroplastik pada Ikan Tongkol Lisong (Auxis Rochei) dan Ikan Tuna Makarel (Euthynnus Affinis) di Pangkalan Pendaratan Ikan (PPI) Oeba, Kupang

Waste management in Kupang City is still very poor. The waste will eventually reach the sea and will be degraded into microplastics. Microplastics, which are less than 5 millimetres in size, are made from plastic waste in marine waters through physical, mechanical, chemical and biological processes. This research was conducted to determine the characteristics (shape and colour) and abundance of microplastics found in digestive tract samples, and meat/muscle samples of lisong tuna (Auxis rochei) and mackerel tuna (Euthynnus affinis) obtained from PPI Oeba, Kupang City. Each organ was extracted using 10% KOH for 48-72 hours and microplastic characteristics were observed visually using a stereo microscope. The research results found microplastics in the digestive tract and meat of A. rochei and E. affinis. The forms found in digestive tract A. rochei include films and fragments, with transparent colours. Meanwhile in meat, fragments, films and fibres were found in red, blue, black, transparent and purple. The forms found in digestive tract E. affinis include fragments, pellets, fibers and films with blue, black, transparent and yellow colours. Meanwhile in meat, fragments and pellets were found with black and yellow colors. The abundance of microplastics detected in digestive tract A. rochei included 1.2 MP/individual, while in meat it was 0.2 MP/gr. In E. affinis it includes 0.8 MP/individual in digestive tract and in meat 0.06 MP/gr.

Leachate analysis of trace metals from e-waste disposed of in landfills and open dumps

Waste electrical and electronic equipment (WEEE, or e-waste) has been largely generated worldwide, but the estimated proportion of WEEE that undergoes the correct disposal is very low. The objective of this study was to evaluate e-waste trace metals in the leachate from disposal of WEEE in soil, using different analytical approaches to determine how these methods may interfere in the results. Different forms of WEEE and MSW landfilling and open dumping were simulated in column reactors to analyse the resultant leachates. Analyses of heavy metals in the leachates were performed in two ways: via inductively coupled plasma-mass-spectrometry with acid digestion of samples, following American Standard 3015A, and via inductively coupled plasma-optical emission spectrometry without acid digestion (US Environmental Protection Agency - USEPA, 2007). Metal concentrations found in the landfill and dumping simulations were similar, but a higher concentration of metals was found in samples with e-waste content, and those undergoing acid digestion prior to ICP analyses. Exceptions were Ba e Zn with a higher concentration without digestion, what may indicate the appearance of matrix effect. Concentrations of Pb, Cu and Mn in the leachates indicate possible interference with MSW. Additionally, it was evident that the timescale of observation influenced the results.

Rapid and simultaneous detection of cadmium and mercury in foods based on solid sampling integrated electrothermal vaporization technique

This work presents an innovative solid sampling (SS) integrated electrothermal vaporization (ETV) approach for simultaneous determination of Cd and Hg based on differentiated elemental vaporization and transportation behavior characteristics. A miniature N2/H2 generator, only consuming electricity and H2O, was utilized to yield reducing atmosphere for Cd vaporization; MgO filler was modified to absorb matrix interferent and keep Hg and Cd transportation via 1st catalytic pyrolysis furnace (CPF); and a gearing was employed to move 2nd CPF to receive and trap (amalgamation) the vaporized Hg from ETV and then thermo-release them for simultaneous detection. Under optimized conditions, the limits of detection of Cd and Hg reached 0.02–0.04 ng/g using 0.4 g sample size. The linearities (R2) exceeded 0.998 and recoveries were 85.0–111.9%, indicating favorable analysis precision and accuracy within ∼3 min without sample digestion process. The proposed HgCd analyzer is suitable for rapid monitoring food with simplicity, green and safety.

Application of microwave digestion for complete dissolution of igneous silicate rock samples: A simple and quick sample preparation procedure

In the area of geochemical analyses of rock solutions, achieving a complete sample dissolution is a fundamental prerequisite for obtaining accurate, precise and reliable analytical results. The challenge posed by the presence of resistant minerals such as zircon, rutile, corundum, spinel, tourmaline, beryl, chromite, and cassiterite in different silicate rocks is a well-recognized challenge in geological studies. These minerals, due to their resilient nature, demand additional efforts to ensure complete dissolution during sample preparation. The prevailing conventional sample digestion methods require several days of laboratory work and the handling of large amounts of multiple types of acids, which also increase sample blanks. Until recently, there was a widely held belief that microwave-assisted digestion, where microwave radiation is transformed to heat, faced limitations in achieving complete dissolution of refractory minerals. This prevailing opinion led to skepticism about the applicability of microwave-assisted digestion for sample preparation of e.g. igneous rock samples containing these minerals. This study introduces a novel, universal and quick closed-vessel (pressurized) high-temperature microwave-assisted digestion method appropriate for dissolution of all major types of igneous silicate rock samples, including rocks containing refractory minerals. This streamlined and expeditious procedure, comprising three steps, requires only a total time of ∼9 h. The method proves its versatility by successfully dissolving both, mafic igneous samples (e.g., basalt) with low-content of resistant minerals, and felsic igneous samples (e.g., granite) with relatively high-content of resistant minerals. To validate the reliability of this procedure, 36 trace elements were analyzed: Li, Be, Sc, V, Cr, Co, Ni, Cu, Zn, Ga, Rb, Sr, Y, Zr, Nb, Cs, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Hf, Ta, Pb, Th and U in several geological Certified Reference Materials (CRMs). The CRMs including basalts JB-3, BCR-2, BHVO-2; andesites JA-2, AGV-2; granodiorite GSP-2; granite JG-2 and alkaline granite MGL-OShBO, were digested and analyzed using triple quadrupole Inductively Coupled-Plasma-Mass Spectrometer (ICP-QQQ). The results of the analysis demonstrate remarkable consistency, closely aligning with both certified and literature values.

Increasing Proteome Coverage Through a Reduction in Analyte Complexity in Single-Cell Equivalent Samples.

The advancement of sophisticated instrumentation in mass spectrometry has catalyzed an in-depth exploration of complex proteomes. This exploration necessitates a nuanced balance in experimental design, particularly between quantitative precision and the enumeration of analytes detected. In bottom-up proteomics, a key challenge is that oversampling of abundant proteins can adversely affect the identification of a diverse array of unique proteins. This issue is especially pronounced in samples with limited analytes, such as small tissue biopsies or single-cell samples. Methods such as depletion and fractionation are suboptimal to reduce oversampling in single cell samples, and other improvements on LC and mass spectrometry technologies and methods have been developed to address the trade-off between precision and enumeration. We demonstrate that by using a monosubstrate protease for proteomic analysis of single-cell equivalent digest samples, an improvement in quantitative accuracy can be achieved, while maintaining high proteome coverage established by trypsin. This improvement is particularly vital for the field of single-cell proteomics, where single-cell samples with limited number of protein copies, especially in the context of low-abundance proteins, can benefit from considering analyte complexity. Considerations about analyte complexity, alongside chromatographic complexity, integration with data acquisition methods, and other factors such as those involving enzyme kinetics, will be crucial in the design of future single-cell workflows.

Peptidomic profile and α-glucosidase inhibitory activity of cooked and gastrointestinal digested legumes

Legumes are a good source of bioactive compounds, including peptides with antidiabetic potential. The α-glucosidase inhibitory activity of simulated gastrointestinal digested (GID) soybean, chickpea, green pea, and navy bean was determined after using three different household cooking methods (conventional, pressure, and microwave cooking). Samples were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC), and the fractions responsible for the α-glucosidase inhibitory activity were isolated. Lastly, peptides were identified by mass spectrometry in tandem (MS/MS) and in silico analyses were done to hypothesise potentially bioactive sequences. The results indicated that all legume extracts exert α-glucosidase inhibitory activity after thermal treatment and GID. Peptide profiles obtained by RP-HPLC showed the highest generation of peptides after the intestinal digestion phase, with small changes between thermal treatments. Best α-glucosidase inhibitory activity was observed in fraction 2 of all gastrointestinal digested samples, with values between 40 % and 62 % inhibition. In soybean, green pea, and navy bean, conventional-cooked samples showed the highest activity, while the pressure-cooked treatment resulted in significantly higher activity in chickpea samples. Finally, 48 peptides were identified by MS/MS and 13 were found as potentially bioactive using in silico tools, expanding previous knowledge on antidiabetic peptides derived from legumes.

Forage Yield and Nutritive Values of Vetches Grown as a Sole and Intercropped with Desho Grass at Different Row Spacing in Western Oromia, Ethiopia

Legume forage, like vetches, is an important feed source for animals as a supplement to low-quality feeds. This study was conducted to evaluate the forage yield and nutritional values of vetches intercropped at various spacings of desho grass. The study was designed in a factorial arrangement with a randomized complete block design with three inter-row spaces (0.50 m, 0.75 m, and 1 m) and intercropping two vetches (<i>Vicia sativa</i> ICARDA 61509 and <i>Vicia dasycarpa</i> lana) and desho grass with three replications. Agronomic parameters, forage and seed yield, chemical analysis, and in vitro digestibility of vetch forage samples were determined. Results showed that significant differences (p<0.05) were observed for all measured agronomic parameters of vetches, except leaf length, due to spacing and interaction effects. Dry matter yield of vetch (DMY) was only affected by row spacing, while crude protein yield (CPY) was affected by row spacing and their interactions. Higher DMY (3.87 - 4.04 t/ha) and CPY (0.74 - 0.79 t/ha) were obtained from solely grown vetches as compared to intercropped vetches (2.26 - 2.42 t/ha and 0.417 - 0.423 t/ha), respectively. Forage yield significantly decreased as row spacing increased. Seed yield decreased as the row space increased, and the yield obtained from <i>Vicia sativa</i> ICARDA 61509 was higher (0.6 and 1.23 t/ha) than <i>Vicia dasycarpa</i> lana (0.41 and 0.82 t/ha) when either intercropped with desho grass or sown alone, respectively. The interaction of intercropping with spacing unaffected vetch's chemical composition. However, crude protein (CP) and acid detergent lignin of vetches were influenced by intercropping, whereas CP, neutral detergent fiber, and acid detergent fiber were affected by spacing (p<0.05). Intercropped <i>Vicia dasycarpa</i> gave higher in vitro dry matter digestibility (IVDMD), <i>in vitro</i> organic matter digestibility, and metabolizable energy than <i>Vicia sativa</i>, and IVDMD increased as row spacing increased from 0.5 to 1 m.

Evaluation of Aflatoxins and Heavy Metals Exposure in the Local Feeds of Fish and Shrimp in Bangladesh.

This study aims to comprehensively assess the presence of aflatoxins, pervasive fungal toxins, and heavy metals (lead, chromium, cadmium) in the local feed of fish and shrimp. Forty-eight samples of the fish and shrimp feed were collected from the Dhaka and Sathkhira districts of Bangladesh respectively. Aflatoxins (B1, B2, G1, G2) were analyzed by high-performance liquid chromatography (HPLC) with fluorescence detector, and atomic absorption spectrophotometry (AAS) was used for the heavy metal detection after sample digestion. The standard analytical methods of the Association of Official Analytical Chemists were followed for analyzing the proximate composition of the feed samples. The findings revealed the high toxic concentration of total aflatoxins particularly in fish feed (196.25 μg/kg). Concurrently, varying concentrations of heavy metals, including lead (Pb) and chromium (Cr), were detected, with the highest levels found in shrimp feed at 3.324 and 174.6 mg/kg, respectively. In comparison, highest contamination of cadmium (Cd) was found in fish feed at 0.398 mg/kg. The outcomes are growing concerns regarding the quality of locally sourced feed and emphasizing the need for prompt corrective actions. Otherwise, neglecting this concern may lead to the possibility of both fish and, ultimately, humans who consume them being vulnerable to the absorption and accumulation of these assessed toxic compounds.

#2000 Analysis between site-specific N-glycosylation of monoclonal immunoglobulin and complement system activation

Abstract Background and Aims Monoclonal immunoglobulin produced by clonal plasma cells is the main cause in multiple myeloma (MM) and monoclonal gammopathy of renal significance (MGRS). Hypocomplementemia and deposition of complement components in the kidney is observed in some patients with MM or MGRS. Previous studies have suggested that glycosylation of the immunoglobulin was associated with complement-mediated-effector activities. Whereas, site-specific N-glycosylation characterization for monoclonal immunoglobulin in MM or MGRS and its effects on complement system activation still remains unclear. Method We separated plasma monoclonal IgG1 and IgA immunoglobulin from patients with MM and human plasma immunoglobulin from healthy controls (HC). Purified IgG and IgA molecules were digested by trypsin. Tryptic peptides without enrichment of intact N-glycopeptides were analyzed using a combination of electron-transfer/higher-energy collisional dissociation (EThcD) and stepped collision energy/higher-energy collisional dissociation (sceHCD) mass spectrometry (EThcD-sceHCD-MS/MS). N-glycosylation characterization was compared between monoclonal immunoglobulin from MM patients and healthy controls and its association with complement level was evaluated. Results A total of 46 IgG1 digestion samples (HC, n = 16; MM, n = 30) and 38 IgA1 digestion samples (HC, n = 16; MM, n = 22) were analyzed using the EThcDsceHCD-MS/MS. On average, 34 and 37 unique N-glycopeptides of IgG1, 19 unique N-glycans per IgG were identified from HC and MM. The relative abundance of fucosylated N-glycans of IgG1 in MM was significantly higher than HC (94.7% vs 88.8%, p = 0.002). Other types of N-glycans including sialylated, hybrid and complex were similar between the two groups. The types of N-glycans including fucosylated, sialylated, hybrid and complex were similar between patients with hypocomplementemia and normal complement levels. Regarding IgA1, on average, 63 and 52 unique N-glycopeptides of IgA1, 40 and 35 unique N-glycans per IgA were identified from HC and MM. The relative abundance of fucosylated (41.0% vs 26.4%, p < 0.001), sialylated (61.0% vs 45.6%, p = 0.003), mannose (2.44% vs 0.28%, p < 0.001) N-glycans of IgA1 in MM was significantly higher than HC. In MM, the relative abundance of mannose N-glycans of IgA1 in patients with hypocomplementemia was significantly lower than patients with normal complement levels (1.69% vs 7.52%, p < 0.001). The relative abundance of fucosylated and sialylated N-glycans were similar between patients with hypocomplementemia and normal complement levels. Conclusion Site-specific N-glycosylation characterization of monoclonal IgG1 and IgA1 in MM patients were differentially expressed from healthy controls. Certain specific N-glycan types were associated with complement levels. These findings laid a foundation for future investigations of their biological effects.

Micro-sample Nd isotopic measurement on reference rock powders via the thermal ionization mass spectrometer Nd+ mode

The Sm–Nd isotopic system has been broadly utilized for dating and tracing in earth and planetary sciences. However, low Nd+ ionization efficiency permanently hinders precise and accurate measurements on small amounts of Nd samples. So far, isotopic measurement of micro-amount Nd sample via thermal ionization mass spectrometry (TIMS) Nd+ mode was mostly performed on Nd reference solutions, but less on natural rock or mineral samples. In the present study, analytical procedures to measure isotopic composition via the TIMS Nd+ mode were designed for natural rock powders by using 1–5 ng pure Nd. The analytical technique was refined through low-blank sample digestion, small-column chemistry for Nd extraction from matrix elements, loading Nd using purified H3PO4 solution, and careful heating of filaments before data acquisition. Measurements on small Nd amounts (1 ng–5 ng) of either the JNdi-1 reference solution or four reference rock powders yielded satisfactory results of 143Nd/144Nd value with respect to precision and accuracy. The analytical results agree well with the recommendation values of the reference materials within analytical errors and the internal precision is better than ±0.005 % (2RSE). When using 1 ng or 2 ng Nd amounts, 144Nd ion flow can remain stable in a signal intensity of >100 mV, and without the assistance of an additional ionization enhancer. Therefore, the analytical procedure, featured by low procedural blank (about 10–22 pg), high Nd+ ionization efficiency, and easy operation, has broad prospects for the application of Nd isotope in single-grain minerals or micro-samples.

Development of New Series of Certified Reference Materials for Ferrosilicon Magnesium Alloys

This paper presents a practical approach to the production of certified reference materials (CRMs) for ferrosilicon magnesium alloys. These new CRMs are predicted to be used in fast X-ray fluorescence spectrometry (XRF) analysis, which does not require sample digestion and does not result in the production of acidic sewage and emissions, contrary to the classical and instrumental techniques currently used in laboratories. Four new certified reference materials (CRMs) were developed to fill the gap in the reference materials market and ensure fast and traceable analyses. The materials were produced with an industrial process and then homogenized and mixed to achieve the required compositions and level of homogeneity. The homogeneity was determined using specially developed analytical methods and confirmed statistically by ANOVA. Additionally, the results of the tests show the short- and long-term stabilities of the new materials. The certified values for specific element contents were determined in interlaboratory tests. All results were assessed statistically for outliers. The results from three or more independent and different analytical methods were used for the calculations. In parallel homogeneity, the stability, and characterization standard uncertainties were calculated and used in the estimation of the final expanded uncertainties of the certified values. Finally, four new CRMs were assisted with the proper certificates according to ISO standards.

Comprehensive Two-Dimensional Liquid Chromatography-High-Resolution Mass Spectrometry for Complex Protein Digest Analysis Using Parallel Gradients.

Despite the high gain in peak capacity, online comprehensive two-dimensional liquid chromatography coupled with high-resolution mass spectrometry (LC × LC-HRMS) has not yet been widely applied to the analysis of complex protein digests. One reason is the method's reduced sensitivity which can be linked to the high flow rates of the second separation dimension (2D). This results in higher dilution factors and the need for flow splitters to couple to ESI-MS. This study reports proof-of-principle results of the development of an RPLC × RPLC-HRMS method using parallel gradients (2D flow rate of 0.7 mL min-1) and its comparison to shifted gradient methods (2D of 1.4 mL min-1) for the analysis of complex digests using HRMS (QExactive-Plus MS). Shifted and parallel gradients resulted in high surface coverage (SC) and effective peak capacity (SC of 0.6226 and 0.7439 and effective peak capacity of 779 and 757 in 60 min). When applied to a cell line digest sample, parallel gradients allowed higher sensitivity (e.g., average MS intensity increased by a factor of 3), allowing for a higher number of identifications (e.g., about 2600 vs 3900 peptides). In addition, reducing the modulation time to 10 s significantly increased the number of MS/MS events that could be performed. When compared to a 1D-RPLC method, parallel RPLC × RPLC-HRMS methods offered a higher separation performance (FHWH from 0.12 to 0.018 min) with limited sensitivity losses resulting in an increase of analyte identifications (e.g., about 6000 vs 7000 peptides and 1500 vs 1990 proteins).