Biological mass spectrometry enables spatiotemporal 'omics: From tissues to cells to organelles.

Biological processes unfold across broad spatial and temporal dimensions, and measurement of the underlying molecular world is essential to their understanding. Interdisciplinary efforts advanced mass spectrometry (MS) into a tour de force for assessing virtually all levels of the molecular architecture, some in exquisite detection sensitivity and scalability in space-time. In this review, we offer vignettes of milestones in technology innovations that ushered sample collection and processing, chemical separation, ionization, and 'omics analyses to progressively finer resolutions in the realms of tissue biopsies and limited cell populations, single cells, and subcellular organelles. Also highlighted are methodologies that empowered the acquisition and analysis of multidimensional MS data sets to reveal proteomes, peptidomes, and metabolomes in ever-deepening coverage in these limited and dynamic specimens. In pursuit of richer knowledge of biological processes, we discuss efforts pioneering the integration of orthogonal approaches from molecular and functional studies, both within and beyond MS. With established and emerging community-wide efforts ensuring scientific rigor and reproducibility, spatiotemporal MS emerged as an exciting and powerful resource to study biological systems in space-time.

Measurement and utilization of the proteomic reactivity by mass spectrometry.

Chemical proteomics, which involves studying the covalent modifications of proteins by small molecules, has significantly contributed to our understanding of protein function and has become an essential tool in drug discovery. Mass spectrometry (MS) is the primary method for identifying and quantifying protein-small molecule adducts. In this review, we discuss various methods for measuring proteomic reactivity using MS and covalent proteomics probes that engage through reactivity-driven and proximity-driven mechanisms. We highlight the applications of these methods and probes in live-cell measurements, drug target identification and validation, and characterizing protein-small molecule interactions. We conclude the review with current developments and future opportunities in the field, providing our perspectives on analytical considerations for MS-based analysis of the proteomic reactivity landscape.

Immunoprecipitation Followed by Mass Spectrometry: An Approach for Identifying Host-Viral Protein-Protein Interactions.

As obligate intracellular parasites, viruses rely on the efficient manipulation of the cell they invade in order to multiply and spread. Protein-protein interactions between viral proteins (or their complexes) and cellular proteins are at the interface between virus and host and hence crucial for the outcome of the infection. Multiple techniques can be used to study protein-protein interactions in vivo in the context of the infected cell; among them, immunoprecipitation followed by mass spectrometry (IP-MS) has proven an efficient approach for the unbiased identification of protein complexes containing a viral protein of interest. In this chapter, we discuss how to employ IP-MS to define the interactome of plant virus proteins by using transient expression in the experimental host Nicotiana benthamiana, using the geminivirus tomato yellow leaf curl virus (TYLCV) as an example.

Mitochondrial phosphoproteomes are functionally specialized across tissues.

Mitochondria are essential organelles whose dysfunction causes human pathologies that often manifest in a tissue-specific manner. Accordingly, mitochondrial fitness depends on versatile proteomes specialized to meet diverse tissue-specific requirements. Increasing evidence suggests that phosphorylation may play an important role in regulating tissue-specific mitochondrial functions and pathophysiology. Building on recent advances in mass spectrometry (MS)-based proteomics, we here quantitatively profile mitochondrial tissue proteomes along with their matching phosphoproteomes. We isolated mitochondria from mouse heart, skeletal muscle, brown adipose tissue, kidney, liver, brain, and spleen by differential centrifugation followed by separation on Percoll gradients and performed high-resolution MS analysis of the proteomes and phosphoproteomes. This in-depth map substantially quantifies known and predicted mitochondrial proteins and provides a resource of core and tissue-specific mitochondrial proteins (mitophos.de). Predicting kinase substrate associations for different mitochondrial compartments indicates tissue-specific regulation at the phosphoproteome level. Illustrating the functional value of our resource, we reproduce mitochondrial phosphorylation events on dynamin-related protein 1 responsible for its mitochondrial recruitment and fission initiation and describe phosphorylation clusters on MIGA2 linked to mitochondrial fusion.

Molecular-level insights into the transformation and degradation pathways of dissolved organic matter during full-scale swine wastewater treatment.

The rapid development of swine farming has resulted in the generation of a large amount of swine wastewater (SW), and dissolved organic matter (DOM) has a crucial role in determining the efficiency and safety of SW treatment. In this study, the transformation and influential mechanisms of DOM on the quality of SW effluent during full-scale SW treatment in actual engineering were systematically investigated using multispectral analysis and the Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) technique. The results showed that S-containing, reduced, saturated, and less aromatic molecules were preferentially removed in the C-AF, while C-S preferentially removed reduced, unsaturated, and aromatic molecules, as well as molecules with large molecular weights. And in the two-stage A/O, the degradation of organic matter and DOM transformation occurred mainly in the A/O-1, with the A/O-2 acting as a supplement to further enhance the humification of DOM. Furthermore, the AOP preferentially removed lignin-like and highly unsaturated compounds, replacing them with a new generation of substances such as proteins and tannins with low aromaticity and unsaturation. More deeply, oxygen addition reactions dominate in both A/O and AOP. Specifically, the most common types of reactions in the A/O were the corresponding potential precursor-product pairs based on methyl to carboxylic acid (-H2 + O2) and alcohol to carboxylic acid (-H2 + O), while tri-hydroxylation (+O3) and di-hydroxylation (+H2O2) reactions were predominant in the AOP. Finally, the study's findings might suggest improving the actual engineering by prioritizing the AOP before the A/O-2 and using the C-S for safeguard treatment of the A/O-2 effluent. It is reliable that this kind of adjustment guarantees safe drainage indications and raises each process unit's efficiency in purifying.

Mass spectrometry-based methods to characterize highly heterogeneous biopharmaceuticals, vaccines, and nonbiological complex drugs at the intact-mass level.

The intact-mass MS measurements are becoming increasingly popular in characterization of a range of biopolymers, especially those of interest to biopharmaceutical industry. However, as the complexity of protein therapeutics and other macromolecular medicines increases, the new challenges arise, one of which is the high levels of structural heterogeneity that are frequently exhibited by such products. The very notion of the molecular mass measurement loses its clear and intuitive meaning when applied to an extremely heterogenous system that cannot be characterized by a unique mass, but instead requires that a mass distribution be considered. Furthermore, convoluted mass distributions frequently give rise to unresolved ionic signal in mass spectra, from which little-to-none meaningful information can be extracted using standard approaches that work well for homogeneous systems. However, a range of technological advances made in the last decade, such as the hyphenation of intact-mass MS measurements with front-end separations, better integration of ion mobility in MS workflows, development of an impressive arsenal of gas-phase ion chemistry tools to supplement MS methods, as well as the revival of the charge detection MS and its triumphant entry into the field of bioanalysis already made impressive contributions towards addressing the structural heterogeneity challenge. An overview of these techniques is accompanied by critical analysis of the strengths and weaknesses of different approaches, and a brief overview of their applications to specific classes of biopharmaceutical products, vaccines, and nonbiological complex drugs.

Radiocarbon (14C) accelerator mass spectrometry as a convenient tool for differentiation of flavor chemicals of synthetic origin from biobased sources and their in-vivo toxicity assessment.

Plants are known to be the natural factory for the production of flavor chemicals. Essential oils comprised of aldehyde as a functional group are potent in deciphering flavor effects in beverages and fresh and prepared food products. In the majority, these are manufactured through synthetic routes, resulting in high product carbon footprints or CO2 equivalents in total greenhouse gas emission. FDA has banned some of the synthetic flavor chemicals due to the health hazards associated with them. However, consumer's preference for natural is at stake due to the absence of quantitative traceability tools. The accelerator mass spectrometer (AMS) analysis revealed a distinction between natural and fossil-derived citral and its blends in Cymbopogon essential oils. The plant-derived citral contained a percent modern carbon (pMC) value in the range of 99-100 %. In contrast, the fossil fuel-derived citral showed zero pMC. Similarly, blends of Cymbopogon oils with 30-50 % (w/w) of fossil origin citral contained pMC equivalent to the proportions of modern carbon. These results showed the usefulness of AMS in quantifying the amount of 14C associated with flavor ingredients. Besides, acute oral toxicity data revealed Cymbopogon oils as the safe flavoring substance at the highest 2000 mg/kg body weight dose in Swiss albino mice.

Tunnel vision in the drinking water research field - Time for non-targeted analysis implementation?

A plethora of compounds can reach our drinking water and possibly affect human health. Still, mostly notorious pollutants like pesticides and disinfection by-products are monitored and regulated. With the increasing availability of high-resolution mass spectrometers (HRMS), non-targeted analyses of environmental samples have become possible. Pilot studies demonstrating the applicability of this approach in the drinking water research field were published. We would like to highlight these studies and appeal to researchers focused on water quality to better exploit the potential of HRMS instruments and broaden the scale of studied pollutants. In addition, the data and experience should be further shared, and the quality standard for the analytical procedures should be set. With advanced knowledge of compounds reaching the drinking water, potential threats would be revealed, and the comprehensive results on water pollution might also act as impulses for associated research branches, including toxicity assessment or development of water treatment technologies, and/or for policy-making.

Measurement of degree of hydrolysis and molecular weight distribution of protein hydrolysates by liquid chromatography-mass spectrometry.

Enzymatic hydrolysis of milk protein is an effective way to improve protein digestibility, to reduce their allergenicity and to produce peptides with better functionalities. Among the process, the degree of hydrolysis (DH) and molecular weight distribution (MWD) of protein hydrolysates are two important parameters that need to be monitored. In this work, a new method based on liquid chromatography-mass spectrometry (LC-MS) was developed for the first time to accurately detect the DH and the MWD of proteolytic peptides. With LC-MS, the content of free amine groups released during hydrolysis was acquired by direct analysis of free trinitrobenzene sulfonic acid (TNBS) for DH assay, overcoming the disadvantage of TNBS-based spectral method. Based on this method, the DH% values of five protein hydrolysis samples were determined and consistent with file specification values. Compared to the size-exclusion high-performance liquid chromatography (SE-HPLC) method, LC-MS was capable of measuring MWD (similar results with file specification values) while additionally providing precise molecular weight and amino acid sequence information for each proteolytic peptide. This method was characterized by its simplicity, accuracy, and reproducibility, making it a valuable technology for monitoring proteolysis and producing peptides with better functionality.

The compatibility rationality of Sijunzi decoction based on integrated analysis of tissue distribution and excretion characteristics in spleen deficiency syndrome rats.

ETHNOPHARMACOLOGICAL RELEVANCE: As a classical prescription for treating spleen deficiency syndrome (SDS), Sijunzi decoction (SJZD) is composed of Ginseng Radix et Rhizoma (RG, Panax ginseng C.A.Mey.), Atractylodes Macrocephalae Rhizoma (AM, Atractylodes macrocephala Koidz.), Poria (Poria cocos (Schw.) Wolf) and Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle (GRP, processed from Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. or Glycyrrhiza glabra L.). The non-polysaccharides (NPSs) are the pharmacodynamic substance basis of SJZD, whose pharmacokinetics in SDS rats were elaborated previously. Further study on their tissue distribution and excretion properties is of significance for understanding the compatibility laws of SJZD. AIM OF THE STUDY: The aim was to unravel the tissue distribution and excretion characteristics of NPSs of SJZD in SDS rats, and explore the scientific connotation of SJZD compatibility. MATERIALS AND METHODS: A validated ultrafast liquid chromatography tandem mass spectrometry method was developed for monitoring the accurate dynamics of sixteen components in the tissues, feces and urine of SDS rats. The four incomplete formulae of SJZD were prepared by randomly deleting one herb to uncover the herb-herb interactions. RESULTS: All components of NPSs in SJZD were distributed in the tissues, except for ononin in the heart. Among them, glycyrrhetinic acid and atractylenolide III were more abundant in the liver and lung, respectively, while other components were enriched in the ileum, especially saponins. The evaluation of fecal excretion and urinary excretion revealed the low cumulative excretion of all components. The comparative analysis of incomplete formulae indicated that the tissue distribution and excretion became faster after removing Poria from SJZD, while a lack of RG led to slower tissue distribution. The tissue distribution at most time points was reduced when AM was absent. Further comprehensive visualization implied that SJZD compatibility can improve tissue distribution of the NPSs, especially ginsenosides and atractylenolide, at the specific time periods. CONCLUSION: The tissue distribution and excretion characteristics of NPSs of SJZD were elucidated in current research. Meanwhile, this study proposed new insights into the mechanism of SJZD compatibility rationality.

Effective multi-stage biodegradation of commercial bulk polyurethane by Clonostachys and Purpureocillium spp.

Dealing with plastic waste in an environmentally friendly and effective manner has been a conundrum that much research has attempted to address. An attractive approach is biodegradation by microorganisms; however microorganisms that have shown such potential are generally only able to degrade surface layers of polymer materials. Herein we describe the multi-stage degradation of commercial bulk polyurethane by two strains of fungi isolated from a landfill. Moreover, we demonstrate that the mechanisms of degradation are not hydrolysis alone as is usually reported, but decarboxylation as well. The data presented here suggest that chain scission at urethane groups involves hydrolysis and decarboxylation of the terminal functional groups and the release of CO2. Two strains, Clonostachys sp. PB54 and Purpureocillium sp. PB57 were both able to decrease the mass of commercial bulk polyurethane by 40 % after 90 days. Spectroscopic analysis revealed the breakage of urethane and ester linkages but also detected variations in hydrogen bonding over time, indicating initial degradation of amorphous surface regions followed by destabilization of more ordered, crystalline layers. Subsequent Liquid Chromatography Mass Spectrometry (LCMS) analysis demonstrated that the fungi were capable of liberating monomer-equivalent molecules (4,4'-methylenedianiline) from the bulk material. In this work, these fungi are shown to be capable of significantly degrading commercial bulk polyurethane in a short period of time, producing small organic molecules and CO2, and as such, are good prospects for the development of large-scale plastic biodegradation processing.

Influence of time-dependent sampling on the plasma metabolome and exposome of fish collected from an effluent-dependent pond.

Metabolomics is increasingly recognized as a useful approach to characterize environmental pollution gradients. While the performance of analytical procedures must be validated and documented, many studies only briefly describe sampling and sample storage. Here we advance our recent study on the influences of sampling delay and holding media on contaminants of emerging concern in fish plasma by targeted analysis. We specifically examined the metabolome and exposome of common carp under three conditions: plasma sampled immediately after field collection (t = 0 h) and then after 3 h (t = 3 h) or 20 h (t = 20 h) of holding fish in lab water. Plasma samples were analyzed using reversed-phase and HILIC chromatography with mass spectrometric detection. 6143 of the 12,904 compounds (after clustering features) varied among the groups. We observed different metabolite variation patterns depending on the sample collection time. We also identified several xenobiotics (2-Ethylhexyl sulfate, 6-Chloro-5-methyl-1H-benzotriazole) at concentrations generally found at the highest levels in plasma sampled immediately after field collection (t = 0 h). Both the metabolome and the exposome changed rapidly in fish plasma with a time lag, which indicates that obtaining relevant results is complicated by fish-holding conditions. We further identified that non-lethal, relatively low-volume blood sample collection was sufficient with this species, which presents ethical and practical advantages.

Liquid-chromatography mass spectrometry describes post-translational modification of Shewanella outer membrane proteins.

Electrogenic bacteria deliver excess respiratory electrons to externally located metal oxide particles and electrodes. The biochemical basis for this process is arguably best understood for species of Shewanella where the integral membrane complex termed MtrCAB is key to electron transfer across the bacterial outer membranes. A crystal structure was recently resolved for MtrCAB from S. baltica OS185. However, X-ray diffraction did not resolve the N-terminal residues so that the lipidation status of proteins in the mature complex was poorly described. Here we report liquid chromatography mass spectrometry revealing the intact mass values for all three proteins in the MtrCAB complexes purified from Shewanella oneidensis MR-1 and S. baltica OS185. The masses of MtrA and MtrB are consistent with both proteins being processed by Signal Peptidase I and covalent attachment of ten c-type hemes to MtrA. The mass of MtrC is most reasonably interpreted as arising from protein processed by Signal Peptidase II to produce a diacylated lipoprotein containing ten c-type hemes. Our two-step protocol for liquid-chromatography mass spectrometry used a reverse phase column to achieve on-column detergent removal prior to gradient protein resolution and elution. We envisage the method will be capable of simultaneously resolving the intact mass values for multiple proteins in other membrane protein complexes.

Quality evaluation of Angelica Sinensis Radix dispensing granules by integrating microvascular activity and chemical analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: As a new form of crude slices of traditional Chinese medicine (TCM), traditional Chinese medicine dispensing granules (TCMDGs) have been used for clinical formula. It is necessary to evaluate whether the chemical composition and biological activity are consistent among the different batches. Angelica Sinensis Radix (ASR), the root of Angelica sinensis (Oliv.) Diels, is one of the most frequently used medicinal materials in gynecology, senile and cardiovascular diseases. In this paper, the quality of TCMDGs is examined taking the Angelica Sinensis Radix dispensing granules (ASRDGs) as a typical case. AIM OF THE STUDY: In this study, integrating bioequivalence and chemical analysis was used to evaluate the quality of dispensing granules taking ASRDGs as a typical case. MATERIALS AND METHODS: According to the clinical efficacy of ASR, the intestinal absorption liquid of ASRDGs (IAL-ASRDGs) in 15 batches prepared by the everted gut sac (EGS) method was used to evaluate its effects of vasodilatation on isolated vascular rings. Then, the chemical profiling analysis of IAL-ASRDGs from the 15 batches was carried out by ultra-high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS). The components in IAL-ASRDGs were identified using mass spectrometry data and the obtained reference standards. Pearson correlation analysis was further performed for the selection of quality control markers based on the extracted ion chromatograms of the identified compounds and vasodilatory activities of different batches of IAL-ASRDGs. Moreover, the selected chemical components in ASRDGs were further verified by vasodilatory activities and quantitatively analyzed by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ-MS). RESULTS: The IAL-ASRDGs showed favorable vasodilatory activities. There were significant differences among the 15 batches. The ranges of maximum vasodilation rate (%) and EC50 were 69.33 ± 7.16 to 19.52 ± 5.05 and 0.07-25.58 g raw materials/mL, respectively. A total of 46 compounds in IAL-ASRDGs were identified based on accurate mass measurements, fragmentation behavior and the reference standards. Among them, 8 compounds including butylidenephthalide, butylphthalide and senkyunolide A showed positive correlation with vasodilatory effect of IAL-ASRDGs. The 8 compounds were further verified, showing obvious vasodilatory activities. The content of the 8 compounds also showed some differences in 15 batches of ASRDGs. Among them, the content of ferulic acid, senkyunolide I and senkyunolide H varied the most in different batches of ASRDGs. By Pearson correlation analysis, the total content of senkyunolides (senkyunolide A, senkyunolide I and senkyunolide H) in ASRDGs was found to reflect the vasodilatory activity of ASRDGs mostly. CONCLUSION: This study provides new strategies for the quality assessment by bioequivalence and explore the chemical quality control markers for ASRDGs.

Development and application of a pseudotargeted lipidomics method for alkylglycerol analysis.

Alkylglycerols (1-O-alkyl-sn-glycerols) are microscale but critical lipids in foods. Conventional lipidomics analysis often loses sight of alkylglycerol analysis. In this study, we developed a high coverage pseudotargeted lipidomics method for analyzing alkylglycerols. The developed method integrated the advantages of GC-MS and LC-MS to profile alkylglycerol-type ether lipids comprehensively, with the help of a data processing Dart package termed FFIMA (Feature Fragments Information Matching Algorithm). The developed method exhibited competitive superiority to conventional lipidomics, such as wider coverage and higher accuracy. The validated method was assessed by three aquatic products and three milks. A total of 25 alkylglycerols, 107 diacylglycerol ethers, 21 monoacylglycerol ethers, 28 alkylglycerol-type ether phospholipids, and 35 plasmalogens were identified in the six foods. The results demonstrated that this method offers a comprehensive analysis of a wide spectrum of alkylglycerols.

B vitamin quantification in lentil seed tissues using ultra-performance liquid chromatography-selected reaction monitoring mass spectrometry.

Lentils are a nutritious food in the human diet. High in protein and with low glycemic index, lentils are also a source of folate and other B vitamins. Understanding variability in B vitamin contents among lentils will allow breeders to select for increased levels. We analyzed 34 cultivated and three wild genotypes for vitamins B1, B2, B3, B5, B6, B7, and B9 in the cotyledons and seed coats. Variation for all B vitamins was observed across the genotypes. Cotyledons had higher concentrations of B1 and B3, while seed coats had higher concentrations of B2, B5, B6, and B9. Wild accessions had the highest concentrations of vitamin B9 and were among the highest for B2. Differential distribution of B vitamins across seed tissues and lentil genotypes has implications for consumption and for breeding. There is useful genetic variability which could be used to increase B vitamin levels in future lentil varieties.

Systematic investigation on the multi-scale mechanisms of bitter peptide self-assembly for flavor modulation.

Suppressing the aversive bitterness of bioactive peptides is an arduous task as it hinders product acceptability. Three acquisition modes (ddMS2, vDIA, and mDIA) of high-resolution mass spectrometry (HRMS) were designed for structure confirmation and accurate quantification of HPFLEWAR, with the mDIA mode chosen as optimum. HRMS and isothermal titration calorimetry was used to elucidate the mechanism that ß-lactoglobulin self-assemble to form association complex in 1:1 stoichiometric ratio (ΔG value - 29.36 kJ mol-1), which automatically attracted HPFLEWAR and reduces its distribution in free form, downgraded the level of bitter perception. Proteomics experiments and molecular dynamics simulations was built to discovered that HPFLEWAR bound and stabilized in the negatively charged region of ß-lactoglobulin via four hydrogen bonds (Lys69, Ile72, Asp53, and Glu74) and hydrophobic interactions. These findings were considered to give theoretical foundation for strictly controlling the bitter perception of peptides and the possible application of HPFLEWAR as new functional components.

The dynamic change of flavor characteristics in Pacific oyster (Crassostrea gigas) during depuration uncovered by mass spectrometry-based metabolomics combined with gas chromatography-ion mobility spectrometry (GC-IMS).

The flavor of Pacific oyster (Crassostrea gigas) significantly changed during the depuration process. This work aimed to explore the mechanism of flavor changes during the 72 h depuration by metabolomics combined with gas chromatography-ion mobility spectrometry (GC-IMS). The metabolomics analysis indicated that carbohydrate metabolism was more affected in the early stage of depuration, including the citrate cycle, glyoxylae and dicarboxylate metabolism, etc. After 72 h depuration, it affected mainly the metabolism of global and overview maps and nucleoside metabolism, etc. The equivalent umami concentration (EUC) value was calculated and exhibited a gradual increase following a 48 h depuration. The GC-MS results revealed that the content of furans was the highest, and the content of aldehydes, ketones, and alcohols was the lowest after 48 h depuration, while the content of aldehydes, ketones, and alcohols increased after 72 h depuration. All these results suggested the depuration period was recommended to be controlled within 48 h.

Standardization of a Mass Spectrometry-Based Workflow for Food Allergen Quantification.

In this chapter, the analytical workflow typically used for the development and validation of an analytical method tailored to food allergen detection and quantification is presented. The main steps defining the workflow are herein described and commented with specific notes about the critical issues that can be faced and common solutions to be adopted. References to guidelines and/or recommendation available from official bodies, as well as main papers from international consortia operating on the specific research field, are also reported, whenever possible. As such, this chapter may represent a practical guide to drive method development in the standardization of analytical methods for food allergen detection.

Fasten the analysis of metal-binding proteins with GE-ICP-MS via increasing the electrolyte concentration of the running buffer.

The coupled system of column gel electrophoresis and inductively coupled plasma mass spectrometry (GE-ICP-MS) is a highly effective technique for detecting metal-binding proteins. However, it takes a long time for this method to test a single sample, which greatly limits its application. In this study, GE-ICP-MS system was optimized by adjusting the analytical conditions, including the concentration and pH of running buffer and the proportion of polyacrylamide gel. The results of the experiment showed that the migration speed of proteins in GE was enhanced by increasing the electrolyte concentration in the running buffer solution. Additionally, the ICP-MS response, which was dramatically decreased because of the change in running buffer solution, can be stabilized by adjusting pH of running buffer. Meanwhile, the optimization of polyacrylamide gel ratio allows GE-ICP-MS to maintain high resolution for proteins of similar molecular weight with increased detection speed. After increasing the concentration of running buffer by 10 times, four iodine labeled proteins were successfully separated at baseline by the GE-ICP-MS system at pH 8.0 in 40 min using a resolving gel (8%, 7 cm) and a stacking gel (4%, 1 cm), which was three times faster than the original one. Finally, the optimized method was proved by detecting a silver-binding protein in rat plasma samples. The above method provided an effective and rapid detection for metal-binding proteins in organism.