Glycoproteomic Reactor for Human Plasma

Zhou, Hu; Hou, Weimin; Denis, Nicholas J.; Zhou, Houjiang; Vasilescu, Julian; Zou, Hanfa; Figeys, Daniel

doi:10.1021/pr800734r

Cited by 29 publications

(42 citation statements)

References 45 publications

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“…Because of the small dimension of these devices, they are readily able to integrate into nanoLC workflows. Various applications have been described including increasing proteome coverage (22,27,28) and targeting of phosphopeptides (24,31,32), glycopeptides and released glycans (29,33,34).…”

mentioning

confidence: 99%

Online Peptide Fractionation Using a Multiphasic Microfluidic Liquid Chromatography Chip Improves Reproducibility and Detection Limits for Quantitation in Discovery and Targeted Proteomics*

Krisp

Yang²,

Soest³

et al. 2015

Molecular & Cellular Proteomics

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Comprehensive proteomic profiling of biological specimens usually requires multidimensional chromatographic peptide fractionation prior to mass spectrometry. However, this approach can suffer from poor reproducibility because of the lack of standardization and automation of the entire workflow, thus compromising performance of quantitative proteomic investigations. To address these variables we developed an online peptide fractionation system comprising a multiphasic liquid chromatography (LC) chip that integrates reversed phase and strong cation exchange chromatography upstream of the mass spectrometer (MS). We showed superiority of this system for standardizing discovery and targeted proteomic workflows using cancer cell lysates and nondepleted human plasma. Five-step multiphase chip LC MS/MS acquisition showed clear advantages over analyses of unfractionated samples by identifying more peptides, consuming less sample and often improving the lower limits of quantitation, all in highly reproducible, automated, online configuration. We further showed that multiphase chip LC fractionation provided a facile means to detect many N-and C-terminal peptides (including acetylated N terminus) that are challenging to identify in complex tryptic peptide matrices because of less favorable ionization characteristics. Given as much as 95% of peptides were detected in only a single salt fraction from cell lysates we exploited this high reproducibility and coupled it with multiple reaction monitoring on a high-resolution MS instrument (MRM-HR). This approach increased target analyte peak area and improved lower limits of quantitation without negatively influencing variance or bias. Further, we showed a strategy to use multiphase LC chip fractionation LC-MS/ MS for ion library generation to integrate with SWATH TM

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mentioning

confidence: 99%

Online Peptide Fractionation Using a Multiphasic Microfluidic Liquid Chromatography Chip Improves Reproducibility and Detection Limits for Quantitation in Discovery and Targeted Proteomics*

Krisp

Yang²,

Soest³

et al. 2015

Molecular & Cellular Proteomics

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“…Several novel glycoproteome analysis systems have been established recently. Zhou et al 28 developed a glycoproteomic reactor that enabled the integration of digestion and deglycosylation. Their approach led to the identification of 82 glycopeptides corresponding to 41 glycoproteins using 5 μL of human serum.…”

mentioning

confidence: 99%

Centrifugation Assisted Microreactor Enables Facile Integration of Trypsin Digestion, Hydrophilic Interaction Chromatography Enrichment, and On-Column Deglycosylation for Rapid and Sensitive N-Glycoproteome Analysis

Zhu¹,

Wang²,

Chen³

et al. 2012

Anal. Chem.

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Sample handling procedures including protein digestion, glycopeptide enrichment, and deglycosylation have significant impact on the performance of glycoproteome analysis. Several glycoproteomic analysis systems were developed to integrate some of these sample preparation procedures. However, no microsystem integrates all of above three procedures together. In this work, we developed a glycoproteomic microreactor enabling seamless integration of all these procedures. In this reactor, trypsin digestion was accelerated by adding acetonitrile to 80%, and after acidification of protein digest by trifluoroacetic acid (TFA), the following hydrophilic interaction chromatography (HILIC) enrichment and deglycosylation were sequentially performed without any desalting, lyophilization, or buffer exchange steps. The total processing time could be as short as 1.5 h. The detection limit of human IgG as low as 30 fmol was also achieved. When applied to human serum glycoproteome analysis, a total number of 92, 178, and 221 unique N-glycosylation sites were identified from three replicate analyses of 10 nL, 100 nL, and 1 μL of human serum, respectively. It was demonstrated that the glycoproteomic microreactor based method had very high sensitivity and was well suited for glycoproteome analysis of minute protein samples.

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“…LC-MS/MS-The LC-MS/MS was performed as previous described (35). Briefly, samples were acidified with formic acid to a final concentration of 5% (v/v) and loaded on a 200 m ϫ 100 mm fused silica precolumn packed in-house with 10 cm of 5-m ReproSil-Pur C18 beads (200 Å; Dr. Maisch GmbH, Ammerbuch, Germany) using an 1100 micro-HPLC system (Agilent Technologies, Santa Clara, CA).…”

Section: Methodsmentioning

confidence: 99%

Improved Recovery and Identification of Membrane Proteins from Rat Hepatic Cells using a Centrifugal Proteomic Reactor

Zhou

Wang

et al. 2011

Molecular & Cellular Proteomics

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Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with >2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism.

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Glycoproteomic Reactor for Human Plasma

Cited by 29 publications

References 45 publications

Online Peptide Fractionation Using a Multiphasic Microfluidic Liquid Chromatography Chip Improves Reproducibility and Detection Limits for Quantitation in Discovery and Targeted Proteomics*

Online Peptide Fractionation Using a Multiphasic Microfluidic Liquid Chromatography Chip Improves Reproducibility and Detection Limits for Quantitation in Discovery and Targeted Proteomics*

Centrifugation Assisted Microreactor Enables Facile Integration of Trypsin Digestion, Hydrophilic Interaction Chromatography Enrichment, and On-Column Deglycosylation for Rapid and Sensitive N-Glycoproteome Analysis

Improved Recovery and Identification of Membrane Proteins from Rat Hepatic Cells using a Centrifugal Proteomic Reactor

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