多壁碳纳米管对质谱分析中的血清蛋白富集作用研究

通过多壁碳纳米管(MWCNTs)对临床血清蛋白提取物进行富集处理,经表面增强激光解析离子化飞行时间质谱(SELDI-TOF-MS)检测,发现MWCNTs对血清中小分子量蛋白(<20 kDa)具有很好的富集效果。同时还考察了内径、长度等参数对血清蛋白富集效果的影响。该方法可用于临床血清样本中低丰度的小分子量蛋白的检测。     

Quantitative analysis of low-abundance serological proteins with peptide affinity-based enrichment and pseudo-multiple reaction monitoring by hybrid quadrupole time-of-flight mass spectrometry

Multiple reaction monitoring (MRM) is commonly used for the quantitative analysis of proteins during mass pectrometry (MS), and has excellent specificity and sensitivity for an analyte in a complex sample. In this study, a pseudo-MRM method for the quantitative analysis of low-abundance serological proteins was developed using hybrid quadrupole time-of-flight (hybrid Q-TOF) MS and peptide affinity-based enrichment. First, a pseudo-MRM-based analysis using hybrid Q-TOF MS was performed for synthetic peptides selected as targets and spiked into tryptic digests of human serum. By integrating multiple transition signals corresponding to fragment ions in the full scan MS/MS spectrum of a precursor ion of the target peptide, a pseudo-MRM MS analysis of the target peptide showed an increased signal-to-noise (S/N) ratio and sensitivity, as well as an improved reproducibility. The pseudo-MRM method was then used for the quantitative analysis of the tryptic peptides of two low-abundance serological proteins, tissue inhibitor of metalloproteinase 1 (TIMP1) and tissue-type protein tyrosine phosphatase kappa (PTPκ), which were prepared with peptide affinity-based enrichment from human serum. Finally, this method was used to detect femtomolar amounts of target peptides derived from TIMP1 and PTPκ, with good coefficients of variation (CV 2.7% and 9.8%, respectively), using a few microliters of human serum from colorectal cancer patients. The results suggest that pseudo-MRM using hybrid Q-TOF MS, combined with peptide affinity-based enrichment, could become a promising alternative for the quantitative analysis of low-abundance target proteins of interest in complex serum samples that avoids protein depletion.     

疏水型色谱饼对人血清蛋白质组学样品的快速分离与制备

建立了疏水型色谱饼(10 mm×20 mm i.d.)与反相色谱(RPLC)离线二维色谱快速分离制备人血清蛋白质组学样品,并用基体辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)进行检测的方法。以4种标准蛋白质的稀溶液为模型进行分离富集,得到细胞色素c(Cyt-c)与肌红蛋白(Myo)的检出限均为1 pmol/μL,溶菌酶(Lys)和胰岛素（Ins）的检出限为0.1 pmol/μL。将此方法用于人血清蛋白质组学样品的分离与制备,随着血清处理量的增大,质谱可检出的组分数目与信号强度均增加,当血清处理量达到1.0 mL时,可检出低丰度蛋白质或多肽285个(相对分子质量均在15000以下)。研究中将1 μg Cyt-c加入到0.5 mL血清中,用上述方法在分离富集低丰度Cyt-c上取得了很好的效果。结果表明,采用疏水型色谱饼与反相色谱联用技术不仅可对血清样品中低丰度蛋白质进行有效的分离和富集,而且一次样品的处理量大,可显著提高低丰度蛋白质的分析、检测水平。     

Hydrazide-functionalized magnetic microspheres for the selective enrichment of digested tryptophan-containing peptides in serum

The extreme complexity of protein samples is becoming a great challenge for proteomic analysis, especially for those having large dynamic range of protein abundance. To solve this problem, and to overcome the limitation of the current proteomic technologies, a new method using hydrazide-functionalized magnetic microspheres was established in this study. With this method, tryptophan (Trp)-containing peptides can be selectively and sensitively enriched from complex and low-volume samples. Furthermore, combined with 1D-LC-MS/MS analysis, the strategy was successfully applied to the proteomic study of mouse serum. The proportion of Trp-containing peptides was increased from 19.4% to 80.2% through enrichment, and the complexity of the sample was reduced more than two times. An additional 113 Trp-containing peptides and 48 novel proteins were detected compared to the conventional method. This enrichment method provides a means for identifying more proteins as potential biomarkers in serum and other complex samples.     

样品制备方法对高效液相色谱-串联质谱分析人乳内源肽的影响

人乳内源肽是乳蛋白在乳腺中被降解形成的具有生理功能的肽,是人乳的重要组成部分,研究人乳内源肽对于婴儿健康具有重要的意义.高效液相色谱-串联质谱(LC-MS/MS)联用技术的应用,促使人乳内源肽的研究取得了突破性的进展.人乳中内源肽含量低、干扰组分多,样品制备方法是影响分析结果的关键步骤.为了研究样品制备方法对分析结果的...     

Enrichment of peptides from plasma for peptidome analysis using multiwalled carbon nanotubes

Human plasma contains a complex matrix of proteolytically derived peptides (plasma peptidome) that may provide a correlate of biological events occurring in the entire organism. Analyzing these peptides from a small amount of serum/ plasma is difficult due to the complexity of the sample and the low levels of these peptides. Here, we describe a novel peptidome analysis approach using multiwalled carbon nanotubes (MWCNTs) as an alternative adsorbent to capture endogenous peptides from human plasma. Harvested peptides were analyzed by using liquid chromatography-mass spectrometry as a means of detecting and assessing the adsorbed molecules. The improved sensitivity and resolution obtained by using liquid chromatography-mass spectrometry allowed detection of 2521 peptide features (m/z 300-1800 range) in about 50 microL of plasma. 374 unique peptides were identified with high confidence by two-dimensional liquid chromatography system coupled to a nano-spray ionization linear ion trap-mass spectrometer. High recovery of BSA digest peptides enriched with MWCNTs, in both standard buffer and high abundance protein solution, was observed. Comparative studies showed that MWCNTs were superior to C18 and C8 for the capture of the smaller peptides. This approach could hold promise of routine plasma peptidome analysis.     

Novel multifunctional chitosan-GMA-IDA-Cu(II) nanospheres for high dynamic range characterization of the human plasma proteome

In this study, we describe characterization of the human plasma proteome based on analysis with multifunctional chitosan-GMA-IDA-Cu(II) nanospheres. Chitosan-GMA-IDA-Cu(II) nanospheres with diameters of 20 to 100?nm have unique properties due to multifunctional chemical moieties, high surface area, high capacity, good dispersibility in buffer solution as well as good biocompatibility and chemical stability which improves their specific interaction with peptides and proteins of the human plasma using different binding buffers. Combining these chitosan-GMA-IDA-Cu(II) nanospheres with MS spectrometry results in a novel strategy which should make it possible to characterize the plasma proteome in a single test. Peptides and proteins adsorbed on the nanosphere can be directly detected by MALDI-TOF-MS. The eluted lower molecular weight peptides and proteins are identified by nano-LC-ESI-MS/MS. A total of 842 unique LMW peptides and 1,682 human unredundant proteins IDs were identified in two different binding buffers, which included relatively low-level proteins (e.g., pg/mL of IL3 Interleukin-3) co-distributed with high-abundance proteins (e.g., 35?C55?mg/mL level serum albumin). As such, this nanosphere technique selectively enabled the identification of proteins over a dynamic range of greater than 8 orders of magnitude. Considering this capacity for selective enrichment of peptides and proteins in human plasma, and the large number of LMW peptides and proteins which can be identified, this method promises to accelerate discovery of biomarkers for clinical application.

Enrichment of peptides in serum by C(8)-functionalized magnetic nanoparticles for direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis

Human serum contains a complex array of proteolytically derived peptides (serum peptidome), which contain biomarkers of preclinical screening and disease diagnosis. Recently, commercial C(8)-functionalized magnetic beads (1-10 microm) were widely applied to the separation and enrichment of peptides in human serum, prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. In this work, laboratory-prepared C(8)-functionalized magnetic nanoparticles (about 50 nm) were prepared and applied to the fast separation and the enrichment of peptides from serum. At first, the C(8)-magnetic nanoparticles were synthesized by modifying amine-functionalized magnetic nanoparticles with chlorodimethyloctylsilane. These synthesized C(8)-amine-functionalized magnetic particles have excellent magnetic responsibility, high dispersibility and large surface area. Finally, the C(8)-magnetic nanoparticles were successfully applied to fast and efficient enrichment of low-abundance peptides from protein tryptic digestion and human serum followed by MALDI-TOF-MS analysis.     

Unmasking low-abundance peptides from human blood plasma and serum samples by a simple and robust two-step precipitation/immunoaffinity enrichment method

Proteomic analysis of human plasma and serum for identifying and validating disease-specific marker proteins and peptides has one major drawback besides its unique advantage as a readily available sample source for diagnostic assays. This disadvantage is represented by the predominance of several high- and middle-abundant proteins, which clearly hamper identification and quantification approaches of potential and validated protein and peptide biomarkers, which are often of very low abundance. During the last decades, a significant number of depletion and enrichment techniques evolved to address these two issues. We present here a cost-effective and easy-to-use strategy for protein depletion comprising a thermal precipitation protocol followed by a two-step liquid/liquid precipitation as well as using an immunoaffinity chromatography method for the specific enrichment and isolation of the low-abundance polypeptide N-terminal pro-B-type natriuretic peptide and its precursor proBNP clinically used as biomarkers for the detection of severe human heart failure and related diseases. The applicability of this approach is shown by SDS -CGE, SDS-PAGE, electrochemiluminescence immunoassay and nano-LC ESI-MS/MS. Our thermal precipitation protocol followed by a two-step liquid/liquid precipitation could also serve as a potential depletion technique for the characterization of other low-abundance peptides and proteins.     

Enhancing plasma peptide MALDI-TOF-MS profiling by mesoporous silica assisted crystallization

Promising profiling techniques based on new material/solid phase extraction for capturing “molecular signatures” from body fluids are being coupled to MALDI-TOF-MS. Sample preparation significantly influences spectrum quality in this ionization method. Mesoporous silica beads (MSB), by the means of nano-sized porous channels with high surface area, enable harvesting of peptides from plasma and serum excluding large size proteins. We have investigated the morphology of a sample slurry, developed as a new tool for plasma peptides enrichment based on mesoporous materials. Our study highlights a correlation between crystals morphology and enhanced performances in MALDI-TOF-MS analysis. This is the first report which correlates the increase in signal intensity with crystal formation in samples preparations which make use of various kinds of slurries for the analysis of samples clinically relevant like human plasma.     

Exosome enrichment of human serum using multiple cycles of centrifugation

In this work, we compared the use of repeated cycles of centrifugation at conventional speeds for enrichment of exosomes from human serum compared to the use of ultracentrifugation (UC). After removal of cells and cell debris, a speed of 110 000 × g or 40 000 × g was used for the UC or centrifugation enrichment process, respectively. The enriched exosomes were analyzed using the bicinchoninic acid assay, 1D gel separation, transmission electron microscopy, Western blotting, and high‐resolution LC‐MS/MS analysis. It was found that a five‐cycle repetition of UC or centrifugation is necessary for successful removal of nonexosomal proteins in the enrichment of exosomes from human serum. More significantly, 5× centrifugation enrichment was found to provide similar or better performance than 5× UC enrichment in terms of enriched exosome protein amount, Western blot band intensity for detection of CD‐63, and numbers of identified exosome‐related proteins and cluster of differentiation (CD) proteins. A total of 478 proteins were identified in the LC‐MS/MS analyses of exosome proteins obtained from 5× UCs and 5× centrifugations including many important CD membrane proteins. The presence of previously reported exosome‐related proteins including key exosome protein markers demonstrates the utility of this method for analysis of proteins in human serum.     

Nanotexture optimization by oxygen plasma of mesoporous silica thin film for enrichment of low molecular weight peptides captured from human serum

This study investigated the optimization of mesoporous silica thin films by nanotexturing using oxygen plasma versus thermal oxidation. Calcination in oxygen plasma provides superior control over pore formation with regard to the pore surface and higher fidelity to the structure of the polymer template. The resulting porous film offers an ideal substrate for the selective partitioning of peptides from complex mixtures. The improved chemico-physical characteristics of porous thin films (pore size distribution, nanostructure, surface properties and pore connectivity) were systematically characterized with XRD, Ellipsometry, FTIR, TEM and N(2) adsorption/desorption. The enrichment of low molecular weight proteins captured from human serum on mesoporous silica thin films fabricated by both methodologies were investigated by comparison of their MALDI-TOF MS profiles. This novel on-chip fractionation technology offers advantages in recovering the low molecular weight peptides from human serum, which has been recognized as an informative resource for early diagnosis of cancer and other diseases.     

Zirconium arsenate-modified silica nanoparticles for specific capture of phosphopeptides and direct analysis by matrix-assisted laser desorption/ionization mass spectrometry

In this paper, we report, as far as we are aware, the first use of zirconium arsenate-modified silica nanoparticles (ZrAs-SNPs) for specific capture of phosphopeptides, followed by matrix-assisted laser desorption/ionization mass spectrometric (MALDI MS) analysis. Under the optimized enrichment conditions, the efficiency and specificity of ZrAs-SNPs were evaluated with tryptic digests of four standard proteins (α-casein, β-casein, ovalbumin, and bovine serum albumin) and compared with those of titanium arsenate-modified silica nanoparticles (TiAs-SNPs). The results showed that more selective enrichment of multiply phosphorylated peptides was observed with ZrAs-SNPs than with TiAs-SNPs whereas TiAs-SNPs resulted in slightly better recovery of singly phosphorylated peptides. ZrAs-SNPs were chosen for direct capture of phosphopeptides from diluted human serum of healthy and adenocarcinoma individuals. Our experimental profiling of serum phosphopeptides revealed that the level of phosphorylated fibrinogen peptide A was up-regulated in the serum of adenocarcinoma patients in comparison with healthy adults. This suggests the possibility of using ZrAs-SNPs for discovery of biomarkers of the pathogenesis process of tumors.     

Highly specific purification of N-glycans using phosphate-based derivatization as an affinity tag in combination with Ti-SPE enrichment for mass spectrometric analysis

N-linked protein glycosylation is involved in regulation of a wide variety of cellular processes and associated with numerous diseases. Highly specific identification of N-glycome remains a challenge while its biological significance is acknowledged. The relatively low abundance of glycan in complex biological mixtures, lack of basic sites for protonation, and suppression by other highly abundant proteins/peptides lead to the particularly poor detection sensitivity of N-glycans in the MS analysis. Therefore, the highly specific purification procedure becomes a crucial step prior to MS analysis of the N-glycome. Herein, a novel N-glycans enrichment approach based on phosphate derivatization combined with Ti⁴⁺-SPE (solid phase extraction) was developed. Briefly, in this strategy, N-glycans were chemically labeled with a phospho-group at their reducing ends, such that the Ti⁴⁺-SPE microspheres were able to capture the phospho-containing glycans. The enrichment method was developed and optimized using model oligosaccharides (maltoheptaose DP7 and sialylated glycan A1) and also glycans from a standard glycoprotein (asialofetuin, ASF). This method experimentally showed high derivatization efficiency (almost 100%), excellent selectivity (analyzing DP7 in the digests of bovine serum albumin at a mass ratio of 1:100), high enriching recovery (90%), good reproducibility (CV<15%) as well as high sensitivity (LOD at fmol level). At last, the proposed method was successfully applied in the profiling of N-glycome in human serum, in which a total of 31 N-glycan masses were identified.     

An investigation into the human serum "interactome"

The protein content of human serum is composed of a millieu of proteins from almost every type of cell and tissue within the body. The serum proteome has been shown to contain information that directly reflects pathophysiological states and represents an invaluable source of diagnostic information for a variety of different diseases. Unfortunately, the dynamic range of protein abundance, ranging from > mg/mL level to < pg/mL level, renders complete characterization of this proteome nearly impossible with current analytical methods. To study low-abundance proteins, which have potential value for clinical diagnosis, the high-abundant species, such as immunoglobulins and albumin, are generally eliminated as the first step in many analytical protocols. This step, however, is hypothesized to concomitantly remove proteins/peptides associated with the high-abundant proteins targeted for depletion. In this study, immunoprecipitation was combined with microcapillary reversed-phase liquid chromatography (microRPLC) coupled on-line with tandem mass spectrometry (MS/MS) to investigate the low-molecular-weight proteins/peptides that associate with the most abundant species in serum. By this targeted isolation of select highly abundant serum proteins, the associated proteins/peptides can be enriched and effectively identified by microRPLC-MS/MS. Among the 210 proteins identified, 73% and 67% were not found in previous studies of the low-molecular-weight or whole-serum proteome, respectively.     

伴刀豆凝集素修饰磁性纳米粒子富集人血清中糖蛋白及质谱鉴定

发展了一种新型的磁性纳米粒子应用于人血清中特异性糖蛋白的亲和富集。制备的磁性纳米粒子具有核/壳/壳结构,即由Fe₃O₄磁性粒子/硅胶层/有机聚合物外层构成。伴刀豆凝集素A（Con A）以共价键合的形式通过短链聚乙二醇固定在粒子表面,实现了人血清中特异性糖蛋白的高效富集。富集的蛋白经过胰蛋白酶酶解后,所得的肽段经离线的二维色谱分离,用高分辨质谱共鉴定出80种蛋白。通过NetNGlyc等搜索软件分析确定其中76种为糖蛋白,分析发现在血清中质量浓度仅为0.00001 g/L的 β -2-glycoprotein 1也得到了鉴定,表明我们发展的磁性纳米粒子与凝集素相结合的方式,可以高效地富集复杂体系中与主要蛋白成分含量相差12个数量级的低丰度糖蛋白。     

Titanium-containing magnetic mesoporous silica spheres: effective enrichment of peptides and simultaneous separation of nonphosphopeptides and phosphopeptides

Protein phosphorylation is a common posttranslational modification, and involved in many cellular processes. Like endogenous peptides, endogenous phosphopeptides contain many biomarkers of preclinical screening and disease diagnosis. In this work, titanium-containing magnetic mesoporous silica spheres were synthesized and applied for effective enrichment of peptides from both tryptic digests of standard proteins and human serum. Besides, the enriched peptides can be further separated into nonphosphopeptides and phosphopeptides by a simple elution. First, titanium-containing magnetic mesoporous silica spheres were synthesized by a sol-gel method and found to have high surface area, narrow pore size distribution, and useful magnetic responsivity. Then, as the prepared material was used for selective capturing of phosphopeptides, it demonstrated to have higher selectivity than commercial titanium dioxide. Moreover, via combination of size-exclusion mechanism, hydrophobic interaction, and affinity chromatography, titanium-containing magnetic mesoporous silica spheres were successfully applied to simultaneously extract and separate nonphosphopeptides and phosphopeptides from standard protein digestion and human serum.     

Preparation of phenyl‐functionalized magnetic mesoporous silica microspheres for the fast separation and selective enrichment of phenyl‐containing peptides

Peptide enrichment before mass spectrometry analysis is essential for large‐scale peptidomic studies, but challenges still remain. Herein, magnetic mesoporous silica microspheres with phenyl group modified interior pore walls were prepared by a facile sol–gel coating strategy, and were successfully applied for selective enrichment of phenyl‐containing peptides in complex biological samples. The newly prepared nanomaterials possessed abundant silanol groups in the exterior surface and numerous phenyl groups in the interior pore walls, as well as a large surface area (592.6 m²/g), large pore volume (0.33 cm³/g), uniform mesopores (3.8 nm), strong magnetic response (29.3 emu/g), and good dispersibility in aqueous solution. As a result of the unique structural properties and size‐exclusion effect, the core–shell phenyl‐functionalized magnetic mesoporous silica microspheres exhibited excellent performance in fast separation and selective enrichment of phenyl‐containing peptides, and the adsorption capacity for bradykinin reached 22.55 mg/g. In addition, selective enrichment of phenyl‐containing peptides from complex samples that are consist of peptides, large proteins, and human serum were achieved by using the as‐prepared microspheres, followed by high‐performance liquid chromatography with ultraviolet detection and electrospray ionization quadrupole time‐of‐flight mass spectrometry analysis. These results demonstrated the as‐prepared microspheres would be a potential candidate for endogenous phenyl‐containing peptides enrichment and biomarkers discovery in peptidome analysis.     

基于共价色谱分离的含半胱氨酸肽段富集策略

多维色谱分离、串联质谱分析技术已在蛋白质组研究中得到广泛应用。然而生物样品的蛋白质以及全酶切肽段具有高度的复杂性,这严重干扰了蛋白质高通量、规模化的分析。通过标签肽段富集进行样品预分离可以降低体系的复杂程度。本文建立了一种基于共价色谱技术选择性分离富集含半胱氨酸肽的方法,从而降低了样品体系的复杂程度。首先以牛血清白蛋白(BSA)的酶切肽段为模型,对富集条件进行了优化和考察,并在此基础上通过5种蛋白质酶切肽段混合物的富集对该方法进行了验证。结果证明此方法的重现性好,富集效率高,富集特异性好,能有效地富集鉴定含半胱氨酸肽段。所建立的方法在复杂体系的蛋白质组研究中具有广泛的应用前景,为复杂样品的蛋白质高通量、自动化、规模化鉴定和定量研究提供了实用技术。     

Selective on-line serum peptide extraction and multidimensional separation by coupling a restricted-access material-based capillary trap column with nanoliquid chromatography–tandem mass spectrometry

As the serum peptidome gets increasing attention for biomarker discovery, one of the important issues is how to efficiently extract the peptides from highly complex human serum for peptidome analysis. Here we developed a fully automated platform for direct injection, on-line extraction, multidimensional separation and MS detection of peptides present in human serum. A capillary SPE column packed with a novel mix mode restricted access material (RAM) exhibiting strong cation exchange and size exclusion chromatography (SCX/SEC) properties were coupled with a nanoliquid chromatography–mass spectrometry (nanoLC-MS) system. The capillary SPE column excludes the high abundant serum proteins such as HSA by size exclusion chromatography and simultaneously extracts the low molecular weight peptides by binding to sulfonic acid residues. Subsequently, the trapped peptides are eluted to a capillary LC column packed with a RP-C18 stationary phase. After injection of only 2 μL human serum to the one-dimensional nanoLC-MS system around 400 peptides could be identified. When conducting a multidimensional separation, the described SCX/SEC/RP-MS platform allows the separation and identification of 1286 peptides present in human serum by the injection and on-line processing of 20 μL human serum sample.