Purpose: The gut microbiome is involved in antitumor immunotherapy and chemotherapy responses; however, evidence-based research on the role of gut microbiome in predicting response to neoadjuvant chemoradiotherapy (nCRT) in patients with locally advanced rectal cancer (LARC) remains scarce. This prospective, longitudinal study aimed to evaluate the feasibility of the gut microbiome in predicting nCRT responses. Experimental Design: We collected 167 fecal samples from 84 patients with LARC before and after nCRT and 31 specimens from healthy individuals for 16S rRNA sequencing. Patients were divided into responders and nonresponders according to pathologic response to nCRT. After identifying microbial biomarkers related to nCRT responses, we constructed a random forest classifier for nCRT response prediction of a training cohort of baseline samples from 37 patients and validated the classifier in another cohort of 47 patients. Results: We observed significant microbiome alterations represented by a decrease in LARC-related pathogens and an increase in Lactobacillus and Streptococcus during nCRT. Furthermore, a prominent microbiota difference between responders and nonresponders was noticed in the baseline samples. Microbes related with butyrate production, including Roseburia, Dorea, and Anaerostipes, were overrepresented in responders, whereas Coriobacteriaceae and Fusobacterium were overrepresented in nonresponders. Ten biomarkers were selected for the response-prediction classifier, including Dorea, Anaerostipes, and Streptococcus, which yielded an area under the curve value of 93.57% [95% confidence interval (CI), 85.76%–100%] in the training cohort and 73.53% (95% CI, 58.96%–88.11%) in the validation cohort. Conclusions: The gut microbiome offers novel potential biomarkers for predicting nCRT responses, which has important manifestations in the clinical management of these patients.

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In regard to the phosphoproteome, highly specific and efficient capture of heteroideous kinds of phosphopeptides from intricate biological sample attaches great significance to comprehensive and in-depth phosphorylated proteomics research. However, until now, it has been a challenge. In this study, a new-fashioned porous immobilized metal ion affinity chromatography (IMAC) material was designed and fabricated to promote the selectivity and detection limit for phosphopeptides by covering a metal-organic frameworks (MOFs) shell onto Fe3O4 nanoparticles, taking advantage of layer-by-layer method (the synthesized nanoparticle denoted as Fe3O4@MIL-100 (Fe)). The thick layer renders the nanoparticles with perfect hydrophilic character, super large surface area, large immobilization of the Fe(3+) ions and the special porous structure. Specifically, the as-synthesized MOF-decorated magnetic nanoparticles own an ultra large surface area which is up to 168.66 m(2) g(-1) as well as two appropriate pore sizes of 1.93 and 3.91 nm with a narrow grain-size distribution and rapid separation under the magnetic circumstance. The unique features vested the synthesized nanoparticles an excellent ability for phosphopeptides enrichment with high selectivity for β-casein (molar ratio of β-casein/BSA, 1:500), large enrichment capacity (60 mg g(-1)), low detection limit (0.5 fmol), excellent phosphopeptides recovery (above 84.47%), fine size-exclusion of high molecular weight proteins, good reusability, and desirable batch-to-batch repeatability. Furthermore, encouraged by the experimental results, we successfully performed the as-prepared porous IMAC nanoparticle in the specific capture of phosphopeptides from the human serum (both the healthy and unhealthy) and nonfat milk, which proves itself to be a good candidate for the enrichment and detection of the low-abundant phosphopeptides from complicated biological samples.

An on-line two-dimensional (2D) capillary electrophoresis (CE) system consisting of capillary isoelectric focusing (CIEF) and capillary gel electrophoresis (CGE) was introduced. To validate this 2D system, a dialysis interface was developed by mounting a hollow fiber on a methacrylate resin plate to hyphenate the two CE modes. The two dimensions of capillary shared a cathode fixated into a reservoir in the methacrylate plate; thus, with three electrodes and only one high-voltage source, a 2D CE framework was successfully established. A practical 2D CIEF-CGE experiment was carried out to deal with a target protein, hemoglobin (Hb). After the Hb variants with different isoelectric points (pIs) were focused in various bands in the first-dimension capillary, they were chemically mobilized one after another and fed to the seconddimension capillary for further separation in polyacrylamide gel. During this procedure, a single CIEF band was separated into several peaks due to different molecular weights. The resulting electrophoregram is quite different from that of either CIEF or CGE; therefore, more information about the studied Hb sample can be obtained.Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) has been a standard technique for high-throughput separation and characterization of biological macromolecules, especially proteins. A main advantage of 2D PAGE is its ability to resolve and investigate the abundance of several thousand proteins in a single sample. 1 More than one sample can be dealt with in a single 2D PAGE run, but a total 2D PAGE process usually lasts for at least 1 day and all the manual operations are time-consuming and laborsome. Capillary electrophoresis (CE) is an easier and more rapid alternative to slab electrophoresis. CE was thoroughly reviewed 2-7 and now is a powerful tool for the separation of proteins and peptides. [8][9][10][11][12][13] Several kinds of CE mode are built based on different principles. Each single CE mode has its distinct capabilities and limitations. To take full advantage of the capabilities and to avoid the limitations of CE modes it is helpful to combine them one to another [14][15][16][17][18][19][20][21][22] or with separation methods based on other than electromigration principles. Much attention bas been paid to the hyphenation of CE with liquid chromatography (LC). [23][24][25][26][27][28][29][30][31] There has been a great deal of effort to interface CE with mass spectrometry (MS); nonetheless, MS was considered as a molecular weight detector rather than a separation tool. [32][33][34][35][36][37][38] * Corresponding author: (e-mail) ykzhang@dicp.ac.cn; (phone) +86-411-3693427; (fax) +86-411-3693427.(1) Lilley, K. S.; Razzaq, A.; Dupree, P. Curr. Opin. Chem. Biol. 2002, 6, 46-50. (2)

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Highly selective and efficient capture of glycosylated proteins and peptides from complex biological samples is of profound significance for the discovery of disease biomarkers in biological systems. Recently, hydrophilic interaction liquid chromatography (HILIC)-based functional materials have been extensively utilized for glycopeptide enrichment. However, the low amount of immobilized hydrophilic groups on the affinity material has limited its specificity, detection sensitivity and binding capacity in the capture of glycopeptides. Herein, a novel affinity material was synthesized to improve the binding capacity and detection sensitivity for glycopeptides by coating a poly(2-(methacryloyloxy)ethyl)-dimethyl-(3-sulfopropyl) ammonium hydroxide (PMSA) shell onto Fe3O4@SiO2 nanoparticles, taking advantage of reflux-precipitation polymerization for the first time (denoted as Fe3O4@SiO2@PMSA). The thick polymer shell endows the nanoparticles with excellent hydrophilic property and several functional groups on the polymer chains. The resulting Fe3O4@SiO2@PMSA demonstrated an outstanding ability for glycopeptide enrichment with high selectivity, extremely high detection sensitivity (0.1 fmol), large binding capacity (100 mg g(-1)), high enrichment recovery (above 73.6%) and rapid magnetic separation. Furthermore, in the analysis of real complicated biological samples, 905 unique N-glycosylation sites from 458 N-glycosylated proteins were reliably identified in three replicate analyses of a 65 μg protein sample extracted from mouse liver, showing the great potential of Fe3O4@SiO2@PMSA in the detection and identification of low-abundance N-linked glycopeptides in biological samples.

Magnetic nanoparticles (MNPs) coated with multilayer polysaccharide shells have been fabricated using a layer-by-layer approach via the alternate deposition of hyaluronan (HA) and chitosan (CS) onto the surface, and the hydrophilic materials were utilized for effective and selective enrichment of glycopeptides in biological samples.Protein glycosylation, one of the most important and ubiquitous post-translation modifications, plays a vital role in regulating various complex biological processes, such as cell-cell interaction, molecular recognition, protein folding, immune response and so forth. 1 Many human diseases have been found to be associated with aberrant N-glycosylation. 2 Currently, mass spectrometry (MS) based techniques have been the premier technology for the characterization of protein glycosylation. Unfortunately, inherent low abundance of glycopeptides, tremendous heterogeneity of each glycosylation site and severe ion suppression caused by the co-existence of non-glycosylated peptides make the direct MS analysis of glycopeptides still a challenge. Hence, an efficient enrichment process prior to MS analysis is needed.To solve these problems, various materials or methods including lectin affinity chromatography, 3a boronic acid affinity chromatography, 3b-d hydrazine beads, 3e Ti(IV)-IMAC 3f and hydrophilic interaction chromatography (HILIC) adsorbents 3g have been utilized for the enrichment of glycopeptides/glycoproteins. Among them, the enrichment strategy based on HILIC has gained increasing popularity and a variety of hydrophilic matrices, such as sepharose, cellulose, ZIC-HILIC beads and click saccharides, have been developed for the selective extraction of glycopeptides. 4 And it was demonstrated that HILIC beads with a high amount of maltose bonded could further improve the profound significance with high selectivity. 5 In recent years, Fe 3 O 4 nanoparticle (MNP) based magnetic separation has become an effective isolation technique, which could achieve better separation compared with conventional approaches, and has attracted much attention in drug delivery, cell separation, bio-separation and enrichment in the biomedical field. 6 Combining the magnetic nano-material with covalently bonded hydrophilic functional molecules could simultaneously achieve the simple and efficient separation of the glycopeptides from the complex peptide mixtures by using magnetic separation. 5,7 Yeh et al. prepared MNPs modified with a hydrophilic polymer by employing spontaneous acid-catalyzed polymerization of zwitterionic monomers, which could be applied for the enrichment of glycopeptides with high selectivity. 7a Recently, a surface initiated atom transfer radical polymerization (SI-ATRP) technique was adopted to obtain the branched PEG brushes hybrid MNPs and then the reactive hydroxyl groups were modified with maltose, which could remarkably increase the amount of immobilized maltose compared to monolayer nanoparticles, and could provide high selectivity and sensitivity for glycopeptide detection. 5...

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