Intestinal extracellular vesicles are altered by vertical sleeve gastrectomy.

Bariatric surgery is the most effective treatment for obesity and its comorbidities. However, our understanding of the molecular mechanisms behind its beneficial effects is limited. Extracellular vesicles (EVs) comprise an important mode of intercellular communication. They carry nucleic acids, hormones, and signaling molecules and regulate multiple processes. Our aim was to test the role of EVs in the effects of vertical sleeve gastrectomy (VSG) using a mouse model. Small intestinal EVs were obtained from the mice that underwent VSG or control surgery and were on chow or high-fat diet or diet-restricted, and then they were subjected to the proteomic analysis. Enteroid and bacterial cultures were treated with EVs to evaluate their survival effect. A mouse cohort received intraduodenal administration of EVs from VSG or Sham mice for 10 days. Body weight, glucose metabolism, and intestinal morphology were evaluated. EVs were enriched in the intestinal lumen and mucus of VSG compared with Sham mice. Protein composition of VSG and Sham-derived EVs was highly distinct. When introduced into culture, VSG EVs decreased survival of intestinal enteroids and, conversely, promoted proliferation of bacteria. Mice administered with EVs obtained from VSG and Sham groups did not show differences in body weight, food intake, or glucose metabolism. Intestinal morphology was altered, as VSG EVs caused reduction of ileal villi length and decreased epithelial proliferation in the jejunum and ileum. VSG causes remodeling of intestinal EVs, which results in unique protein composition. VSG-derived EVs exhibit cytotoxic effects on epithelial cells and reduce proliferation of intestinal progenitor cells in mice.NEW & NOTEWORTHY This is the first study that investigates the impact of bariatric surgery on protein composition of intestinal extracellular vesicles. Extracellular vesicle composition is greatly altered after vertical sleeve gastrectomy and may potentially modulate various signaling pathways. In our study, extracellular vesicles from vertical sleeve gastrectomy-treated mice promote bacterial proliferation but exhibit cytotoxic effect on epithelial cells and reduce proliferation of intestinal progenitor cells in mice.

Differential Quantitative Determination of Site-Specific Intact N-Glycopeptides in Serum Haptoglobin between Hepatocellular Carcinoma and Cirrhosis Using LC-EThcD-MS/MS.

Intact N-glycopeptide analysis remains challenging due to the complexity of glycopeptide structures, low abundance of glycopeptides in protein digests, and difficulties in data interpretation/quantitation. Herein, we developed a workflow that involved advanced methodologies, the EThcD-MS/MS fragmentation method and data interpretation software, for differential analysis of the microheterogeneity of site-specific intact N-glycopeptides of serum haptoglobin between early hepatocellular carcinoma (HCC) and liver cirrhosis. Haptoglobin was immunopurified from 20 µL of serum in patients with early HCC, liver cirrhosis, and healthy controls, respectively, followed by trypsin/GluC digestion, glycopeptide enrichment, and LC-EThcD-MS/MS analysis. Identification and differential quantitation of site-specific N-glycopeptides were performed using a combination of Byonic and Byologic software. In total, 93, 87, and 68 site-specific N-glycopeptides were identified in early HCC, liver cirrhosis, and healthy controls, respectively, with high confidence. The increased variety of N-glycopeptides in liver diseases compared to healthy controls was due to increased branching with hyper-fucosylation and sialylation. Differential quantitation analysis showed that 5 site-specific N-glycopeptides on sites N184 and N241 were significantly elevated in early HCC compared to cirrhosis ( p < 0.05) and normal controls ( p ≤ 0.001). The result demonstrates that the workflow provides a strategy for detailed profiles of N-glycopeptides of patient samples as well as for relative quantitation to determine the level changes in site-specific N-glycopeptides between disease states.

Comparison of an Optimized Ultracentrifugation Method versus Size-Exclusion Chromatography for Isolation of Exosomes from Human Serum.

Exosomes are nanosized vesicles that are abundant in biological fluids. In recent years, exosomes have attracted increasing attention as their cargo may provide promising biomarkers for the early diagnosis of and therapy for many diseases, such as cancer. In addition to ultracentrifugation (UC), many alternative methods including size-exclusion chromatography (SEC) have been developed for isolating exosomes. It has been reported that the SEC method provided improved performance relative to the UC method in isolating exosomes from plasma, where the former contained less residual blood protein contamination. We have compared the SEC method with an optimized UC method in isolating exosomes from human serum. This was based on dilution of the serum to reduce the viscosity and a prolonged cycle of UC, followed by another four cycles. We found that >95% of serum proteins were removed without a significant loss of exosome proteins relative to SEC. We also combined one cycle of UC with SEC and found that this method provided improved results relative to the SEC method, although the serum protein contamination was several times higher than that of our optimized UC method. The TEM showed that the size distribution of exosomes isolated from each of the three methods was similar.

Circulating Microvesicles from Pancreatic Cancer Accelerate the Migration and Proliferation of PANC-1 Cells.

Circulating microvesicles are able to mediate long-distance cell-cell communications. It is essential to understand how microvesicles from pancreatic cancer act on other cells in the body. In this work, serum-derived microvesicles were isolated from 10 patients with locally advanced pancreatic cancer and healthy controls. Using Cell Transwell and WST-1 reagents, we found that microvesicles from pancreatic cancer accelerated migration and proliferation of PANC-1 cells. Meanwhile, the proliferation of these cancer-microvesicle-treated cells (CMTCs) was affected less by 10 µM of gemcitabine relative to healthy microvesicle-treated cells (HMTCs). Next, we optimized the filter-aided sample preparation method to increase the recovery of protein samples and then applied it to the quantification of the proteome of CMTCs and HMTCs. The peptides were labeled and analyzed by liquid chromatography-tandem mass spectrometry. In total, 4102 proteins were identified, where 35 proteins were up-regulated with 27 down-regulated in CMTCs. We verified the quantitative results of three key proteins CD44, PPP2R1A, and TP53 by Western blot. The Ingenuity Pathway Analysis revealed pathways that cancer microvesicles might participate in to promote cell migration and proliferation. These findings may provide novel clues of treatment for tumorigenesis and metastasis.

High-Performance Chemical Isotope Labeling Liquid Chromatography Mass Spectrometry for Exosome Metabolomics.

Circulating exosomes in bodily fluids such as blood are being actively studied as a rich source of chemical biomarkers for cancer diagnosis and monitoring. Although nucleic acid analysis is a primary tool for the discovery of circulating biomarkers in exosomes, metabolomics holds the potential of expanding the chemical diversity of biomarkers that may be easy and rapid to detect. However, only trace amounts of exosomes can be isolated from a small volume of patient blood, and thus a very sensitive technique is required to analyze the metabolome of exosomes. In this report, we present a workflow that involves multiple cycles of ultracentrifugation for exosome isolation using a starting material of 2 mL of human serum, freeze-thaw-cycles in 50% methanol/water for exosome lysis and metabolite extraction, differential chemical isotope labeling (CIL) of metabolites for enhancing liquid chromatography (LC) separation and improving mass spectrometry (MS) detection, and nanoflow LC-MS (nLC-MS) with captivespray for analysis. As a proof-of-principle, we used dansylation labeling to analyze the amine- and phenol-submetabolomes in two sets of exosome samples isolated from the blood samples of five pancreatic cancer patients before and after chemotherapy treatment. The average number of peak pairs or metabolites detected was 1964 ± 60 per sample for a total of 2446 peak pairs ( n = 10) in the first set and 1948 ± 117 per sample for a total of 2511 peak pairs ( n = 10) in the second set. There were 101 and 94 metabolites positively identified in the first and second set, respectively, and 1580 and 1590 peak pairs with accurate masses matching those of metabolites in the MyCompoundID metabolome database. Analyzing the mixtures of 12C-labeled individual exosome samples spiked with a 13C-labeled pooled sample which served as an internal standard allowed relative quantification of metabolomic changes of exosomes of blood samples collected before and after treatment.

The analysis of alpha-1-antitrypsin glycosylation with direct LC-MS/MS.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methodology has been developed to differentiate core- and antennary-fucosylated glycosylation of glycopeptides. Both the glycosylation sites (heterogeneity) and multiple possible glycan occupancy at each site (microheterogeneity) can be resolved via intact glycopeptide analysis. The serum glycoprotein alpha-1-antitrypsin (A1AT) which contains both core- and antennary-fucosylated glycosites was used in this study. Sialidase was used to remove the sialic acids in order to simplify the glycosylation microheterogeneity and to enhance the MS signal of glycopeptides with similar glycan structures. ß1-3,4 galactosidase was used to differentiate core- and antennary-fucosylation. In-source dissociation was found to severely affect the identification and quantification of glycopeptides with low abundance glycan modification. The settings of the mass spectrometer were therefore optimized to minimize the in-source dissociation. A three-step mass spectrometry fragmentation strategy was used for glycopeptide identification, facilitated by pGlyco software annotation and manual checking. The collision energy used for initial glycopeptide fragmentation was found to be crucial for improved detection of oxonium ions and better selection of Y1 ion (peptide+GlcNAc). Structural assignments revealed that all three glycosylation sites of A1AT glycopeptides contain complex N-glycan structures: site Asn70 contains biantennary glycans without fucosylation; site Asn107 contains bi-, tri- and tetra-antennary glycans with both core- and antennary-fucosylation; site Asn271 contains bi- and tri-antennary glycans with both core- and antennary-fucosylation. The relative intensity of core- and antennary-fucosylation on Asn107 was similar to that of the A1AT protein indicating that the glycosylation level of Asn107 is much larger than the other two sites.

Quantitative Proteomic Analysis of Serum Exosomes from Patients with Locally Advanced Pancreatic Cancer Undergoing Chemoradiotherapy.

Pancreatic cancer is the third leading cause of cancer-related death in the USA. Despite extensive research, minimal improvements in patient outcomes have been achieved. Early identification of treatment response and metastasis would be valuable to determine the appropriate therapeutic course for patients. In this work, we isolated exosomes from the serum of 10 patients with locally advanced pancreatic cancer at serial time points over a course of therapy, and quantitative analysis was performed using the iTRAQ method. We detected approximately 700-800 exosomal proteins per sample, several of which have been implicated in metastasis and treatment resistance. We compared the exosomal proteome of patients at different time points during treatment to healthy controls and identified eight proteins that show global treatment-specific changes. We then tested the effect of patient-derived exosomes on the migration of tumor cells and found that patient-derived exosomes, but not healthy controls, induce cell migration, supporting their role in metastasis. Our data show that exosomes can be reliably extracted from patient serum and analyzed for protein content. The differential loading of exosomes during a course of therapy suggests that exosomes may provide novel insights into the development of treatment resistance and metastasis.

Influence of different processing times on the quality of Polygoni Multiflora Radix by metabolomics based on ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry.

A metabolomics method based on ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry was developed to evaluate the influence of processing times on the quality of raw and processed Polygoni Multiflora Radix. Principal component analysis and partial least-squares discriminant analysis was used to screen the potential maker metabolites that were contributed to the quality changes. Then these marker metabolites were selected as variables in Fisher's discriminant analysis to establish the models that were used to distinguish the raw and processed Polygoni Multiflora Radix in the markets. Additionally, 36 compounds were identified. Twelve raw Polygoni Multiflora Radix samples and 23 processed Polygoni Multiflora Radix samples were distinguished. The results showed that the 12 raw Polygoni Multiflora Radix samples belonged to the group of processing time of 0 h, and two processed Polygoni Multiflora Radix samples were part of the group of processing times of 4 h, 12 samples belonged to group of processing times of 8 to 16 h, and nine samples were the group of processing times of 24 to 48 h. The results demonstrated that the method could provide scientific support for the processing standardization of Polygoni Multiflora Radix.

Development of an Integrated Pipeline for Profiling Microbial Proteins from Mouse Fecal Samples by LC-MS/MS.

Metaproteomics is one approach to analyze the functional capacity of the gut microbiome but is limited by the ability to evenly extract proteins from diverse organisms within the gut. Herein, we have developed a pipeline to optimize sample preparation of stool obtained from germ-free (GF) mice that were gavaged a defined community of 11 bacterial strains isolated from the human gut. With 64% more proteins identified, bead-beating was confirmed to be an indispensable step for the extraction of bacterial proteins, especially for Gram-positive bacteria. Bacterial enrichment from mouse fecal samples was further optimized by evaluating three different methods: (1) a high-speed differential centrifugation (HCE) or (2) a low-speed differential centrifugation (LCE) and (3) a filter-aided method (FA). The HCE method was associated with dramatic loss of bacteria and 71% less recovery of bacterial proteins than the LCE method. Compared with LCE, the FA method also showed dramatic loss of the amount of bacteria recovered and decreased protein identifications from Gram-positive bacteria in the stool samples. Ultimately, LCE may provide an alternative and complementary method for enriching bacteria from small amounts of mouse fecal samples, which could aid in investigating bacterial function in health and disease.

A procedure for the analysis of site-specific and structure-specific fucosylation in alpha-1-antitrypsin.

A MS-based methodology has been developed for analysis of core-fucosylated versus antennary-fucosylated glycosites in glycoproteins. This procedure is applied to the glycoprotein alpha-1-antitrypsin (A1AT), which contains both core- and antennary-fucosylated glycosites. The workflow involves digestion of intact glycoproteins into glycopeptides, followed by double digestion with sialidase and galactosidase. The resulting glycopeptides with truncated glycans were separated using an off-line HILIC (hydrophilic interaction liquid chromatography) separation where multiple fractions were collected at various time intervals. The glycopeptides in each fraction were treated with PNGase F and then divided into halves. One half of the sample was applied for peptide identification while the other half was processed for glycan analysis by derivatizing with a meladrazine reagent followed by MS analysis. This procedure provided site-specific identification of glycosylation sites and the ability to distinguish core fucosylation and antennary fucosylation via a double digestion and a mass profile scan. Both core and antennary fucosylation are shown to be present on various glycosites in A1AT.

Trace amounts of poly-ß-cyclodextrin wrapped carbon nanotubes for the microextraction of flavonoids in honey samples by capillary electrophoresis with light-emitting diode induced fluorescence detection.

A novel dispersive micro-SPE method with trace poly-ß-CD wrapped multiwalled carbon nanotubes as sorbents was applied to extract flavonoids in honey samples. The analytes were then determined by CE with LED-induced fluorescence detection. The influencing parameters, such as the sorbent concentration, extraction time, and eluent type, were properly optimized. The established method had the advantages of simplicity, low consumption of sorbent amount (0.009 mg) and organic solvent (100 µL), and high sensitivity, meeting the principle of green chemistry. Under the optimum conditions, the sorbents allowed the extraction and preconcentration of the flavonoids with enrichment factors in the range from 78 to 166. The recovery study performed at two different fortification levels provided an average recovery >91%. LODs for all the compounds ranged from 0.07 to 17.99 ng/mL. These results demonstrated that the proposed method could be used as a convenient and efficient extraction technique for analysis of flavonoids in different honey matrices.

Analysis of isoquinoline alkaloids using chitosan-assisted liquid-solid extraction followed by microemulsion liquid chromatography employing a sub-2-micron particle stationary phase.

A simple, efficient, and green chitosan-assisted liquid-solid extraction method was developed for the sample preparation of isoquinoline derivative alkaloids followed by microemulsion LC. The optimized mobile phase consisted of 0.8% w/v of ethyl acetate, 1.0% w/v of SDS, 8.0% w/v of n-butanol, 0.1% v/v acetic acid, and 10% v/v ACN. Compared to pharmacopoeia method and organic solvent extraction, this new approach avoided the use of volatile organic solvents, replacing them with relatively small amounts of chitosan. Under the optimum conditions, good linearity (r2 > 0.9980) for all calibration curves and low detection limits between 0.05 and 0.10 µg/mL were achieved. The presented procedure was successfully applied to determine alkaloids in Rhizoma coptidis with satisfactory recoveries (81.3-106.4%).

The alteration of H4-K16ac and H3-K27met influences the differentiation of neural stem cells.

The neural stem cell therapy provides a promising future for patients with central nerve system damage, thus an insight into its differentiation mechanism is urgently needed. Herein, we aimed to identify various histone modifications and reveal their impact on the differentiation of neural stem cells (NSCs) toward neurons. Firstly, we labeled primary NSCs using the stable isotope labeling with amino acids in cell culture (SILAC) technique. Then we induced these NSCs to differentiate by all-trans retinoic acid (atRA) or SB216763. Next, we identified the alteration of histone modifications in early-differentiated NSCs by mass spectrometry and verified them by Western blot. Interestingly, these modification alterations and phenotype changes were found similar in NSCs induced by the two different drugs. More interestingly, during the differentiation process H3-K27met was significantly up-regulated while H4-K16ac was not altered at the global level but down-regulated in some low-abundance combinatorial codes. We inhibited the methyltransferase of H3-K27 and deacetylase of H4-K16 simultaneously and found the differentiation procedure was obviously delayed. The function of H4-K16ac and H3-K27met in NSCs differentiation would be useful to reveal the differentiation mechanism and valuable for further neural stem cell therapy.

A Review on Phytochemistry and Pharmacology of Cortex Periplocae.

Cortex Periplocae, as a traditional Chinese herbal medicine, has been widely used for autoimmune diseases, especially rheumatoid arthritis. Due to its potential pharmaceutical values, more studies about the biological activities of Cortex Periplocae have been conducted recently. Meanwhile, the adverse reaction of Cortex Periplocae is not a negligible problem in clinic. In this article, we reviewed a series of articles and summarized the recent studies of Cortex Periplocae in the areas of phytochemistry and pharmacology. More than 100 constituents have been isolated and identified from Cortex Periplocae, including steroids, cardiac glycosides, terpenoids, and fatty acid compounds. The crude extracts of Cortex Periplocae and its active compounds exhibit various biological activities, such as cardiotonic effect, anticancer action, and anti-inflammatory effect. This paper aims to provide an overall review on the bioactive ingredients, pharmacological effect, and toxicity of this plant. Furthermore, this review suggests investigating and developing new clinical usages according to the above pharmacological effects.

Evaluation of the accuracy of protein quantification using isotope TMPP-labeled peptides.

N-succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl) phosphonium bromide (TMPP-Ac-OSu) reacts rapidly, mildly, and specifically with the N-terminals of proteins and peptides. Thus, it can be developed as an ideal isotope-coded tag to be used in quantitative proteomics. Here, we present a strategy for light and heavy TMPP-based quantitative proteomic analysis, in which peptides in a mixture can be quantified using an on-tip TMPP derivatization approach. To demonstrate the accuracy of this strategy, light and heavy TMPP-labeled peptides were combined at different ratios and subsequently analyzed by LC-MS/MS. The MS spectra and scatter plots show that peptide and protein ratios were both consistent with the mixed ratios. We observed a linear correlation between protein ratios and the predicted ratios. In comparison with SILAC method, the TMPP labeling method produced similarly accurate quantitative results with low CVs. In conclusion, our results suggest that this isotope-coded TMPP method achieved accurate quantification and compatibility with IEF-based separation. With the inherent advantages of TMPP derivatization, we believe that it holds great promise for future applications in quantitative proteomics analysis.

Citric acid-assisted two-step enrichment with TiO2 enhances the separation of multi- and monophosphorylated peptides and increases phosphoprotein profiling.

Phosphopeptide enrichment is essential for large-scale phosphoprotein profiling. Titanium dioxide (TiO2) is widely used in phosphopeptide enrichment, but it is limited in the isolation of multiphosphorylated peptides due to their strong binding. In this study, we found that citric acid greatly affects the binding of mono- and multiphosphopeptides with TiO2, which can be used for stepwise phosphopeptide separation coupled with mass spectrum (MS) identification. We first loaded approximately 1 mg of peptide mixture of HeLa cell digests onto TiO2 beads in highly concentrated citric acid (1 M). Then the flow-through fraction was diluted to ensure low concentration of citric acid (50 mM) and followed by loading onto another aliquot of TiO2 beads. The two eluted fractions were subjected to nanoLC-MS/MS analysis. We identified 1,500 phosphorylated peptides, of which 69% were multiphosphorylated after the first enrichment. After the second enrichment, 2,167 phosphopeptides, of which 92% were monophosphorylated, were identified. In total, we successfully identified 3,136 unique phosphopeptides containing 3,973 phosphosites utilizing this strategy. Finally, more than 37% of the total phosphopeptides and 2.6-fold more of the multiphosphorylated peptides were identified as compared to the frequently used DHB/TiO2 enrichment strategy. Combining SCX with CATSET, we identified 14,783 phosphopeptides and 15,713 phosphosites, of which 3,678 were unrecorded in PhosphoSitePlus database. This two-step separation procedure for sequentially enriching multi- and monophosphorylated peptides by using citric acid is advantageous in multiphosphorylated peptide separation, as well as for more comprehensive phosphoprotein profiling.

High-confidence de novo peptide sequencing using positive charge derivatization and tandem MS spectra merging.

De novo peptide sequencing holds great promise in discovering new protein sequences and modifications but has often been hindered by low success rate of mass spectra interpretation, mainly due to the diversity of fragment ion types and insufficient information for each ion series. Here, we describe a novel methodology that combines highly efficient on-tip charge derivatization and tandem MS spectra merging, which greatly boosts the performance of interpretation. TMPP-Ac-OSu (succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide) was used to derivatize peptides at N-termini on tips to reduce mass spectra complexity. Then, a novel approach of spectra merging was adopted to combine the benefits of collision-induced dissociation (CID) and electron transfer dissociation (ETD) fragmentation. We applied this methodology to rat C6 glioma cells and the Cyprinus carpio and searched the resulting peptide sequences against the protein database. Then, we achieved thousands of high-confidence peptide sequences, a level that conventional de novo sequencing methods could not reach. Next, we identified dozens of novel peptide sequences by homology searching of sequences that were fully backbone covered but unmatched during the database search. Furthermore, we randomly chose 34 sequences discovered in rat C6 cells and verified them. Finally, we conclude that this novel methodology that combines on-tip positive charge derivatization and tandem MS spectra merging will greatly facilitate the discovery of novel proteins and the proteome analysis of nonmodel organisms.

Sequential Method for Analysis of CTCs and Exosomes from the Same Sample of Patient Blood.

Circulating tumor cells (CTCs) and exosomes, both released from the primary tumor into peripheral blood, are a promising source of cancer biomarkers. They are detectable in the blood and carry a large diversity of biological molecules, which can be used for the diagnosis and monitoring of minimally invasive cancers. However, due to their intrinsic differences in counts, size, and molecular contents, studies have focused on only one type of vesicle. Herein, we have developed an integrated system to sequentially isolate CTCs and exosomes from a single patient blood sample for further profiling and analysis. The CTCs are isolated using a commercial filtration method and then the remaining blood is processed using multiple cycles of ultracentrifugation to isolate the exosomes. The method uses two available technologies where the eluent from CTC isolation is usually discarded and interfaces them, so that the eluent can be interfaced to exosome isolation methods. The CTCs are identified based on fluorescence staining of their surface markers, while the exosomes are analyzed using transmission electron microscopy, nanosight tracking analysis, and mass spec proteomic analysis. This analysis showed CTCs detected by their surface markers for metastatic hepatocellular carcinoma (HCC), while essentially none were detected for cirrhosis. The exosome analysis resulted in the identification of ∼500-1000 exosome proteins per sample confirmed by detection of exosome surface markers CD9, CD63, CD81, and TSG101 in addition to proteins related to cancer progression. Proteins enriched in HCC exosomes were shown to be involved in the immune response, metastasis, and proliferation.

Comprehensive analysis of sequence-specific stability of siRNA.

Double-stranded small interfering RNAs (siRNAs) are important modulators of biological processes and hold great promise for therapeutic applications. However, serum processing of synthetic siRNAs is still largely unknown. To address this issue, serum degradation assays of 125 siRNAs were first performed in this study. Four siRNA categories of distinct serum stability were identified, including a group of siRNAs that were stable in their native form for both in vitro and in vivo assays. Fine mapping of the cleavage events occurring in serum treatment demonstrated that most occurred at two vulnerable sites, leading to a speculation that rational modification of these sites might protect most siRNAs from serum degradation. For proof of concept, an exhaustive siRNA modification study was performed. In addition to the consistent stabilization pattern revealed at these sites, our study further showed that a single modification made at the cleavage site stabilized the siRNAs to a large extent, highlighting the importance of these sites in siRNA degradation. In summary, the present study provided a comprehensive picture of serum processing of siRNA as well as a starting point for a rational siRNA modification strategy, both of which are of great importance to in vivo and therapeutic applications of siRNA.

Enrichment of serum low-molecular-weight proteins using C18 absorbent under urea/dithiothreitol denatured environment.

Serum low-molecular-weight proteins (LMWPs, molecular weight<30kDa) are closely related to the body physiological and pathological situations, whereas many difficulties are encountered when enriching and fractionating them. Using C(18) absorbent (100 A) enrichment and fractionation under urea/dithiothreitol (DTT) denatured environment followed by 60% acetonitrile (ACN) elution, serum LMWPs could be enriched more than 100-fold and were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE), and isotope-coded affinity tag (ICAT) labeling quantification. Proteins existing in human serum at low nanograms/milliliter (ng/ml) levels, such as myeloid-related proteins (MRPs), could be identified directly from 2-DE coupled with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and LTQ-Orbitrap MS. Sixteen proteins were confidentially identified and quantified using ICAT labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS). By virtue of its easy operation and high reproducibility to process large quantity complex serum samples, this method has potential uses in enriching LMWPs either in serum or in cell and tissue samples.