Human Plasma Proteome Analysis by Multidimensional Chromatography Prefractionation and Linear Ion Trap Mass Spectrometry Identification

Jin, Wenhai; Dai, Jie; Li, Su-Jun; Xia, Qi-Chang; Zou, Hanfa; Zeng, Rong

doi:10.1021/pr049761h

Cited by 76 publications

(67 citation statements)

References 34 publications

(54 reference statements)

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“…There are several factors that influence the number of proteins identified in any proteomic study: (i) quantity of sample, (ii) extent of fractionation, (iii) loading capacity of the technology, and (iv) the stringency of criteria used for the protein identification. In a recent study, peptide fragments obtained from digestion of 1.1 ml (Ͼ50 mg of protein) of undepleted plasma were fractionated using ion exchange chromatography followed by capillary reversed phase LC (12). The authors reported the identities of 1292 non-redundant proteins.…”

Section: Table II Optimization Of Chromatographic Focusing Conditionsmentioning

confidence: 99%

Multidimensional Liquid Chromatography Separation of Intact Proteins by Chromatographic Focusing and Reversed Phase of the Human Serum Proteome

Sheng

Chen

Eyk

2006

Molecular & Cellular Proteomics

102

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In biomarker discovery, the detection of proteins with low abundance in the serum proteome can be achieved by optimization of protein separation methods as well as selective depletion of the higher abundance proteins such as immunoglobins (e.g. IgG) and albumin. A relative newcomer to the proteomic separation arena is the commercial instrument PF2D from Beckman Coulter that separates proteins in the first dimension using chromatofocusing followed in line by reversed phase chromatography in the second dimension, thereby separating intact proteins based on pI and hydrophobicity. In this study, assessment and optimization of serum separation (undepleted serum and albumin-IgG-depleted serum) by the PF2D is presented. Protein databases were created for serum obtained from a healthy individual under traditional and optimized methods and under different sample preparation protocols. Separation of the doubly depleted serum using the PF2D with 20% isopropanol present in the first dimension running buffer allowed us to unambiguously identify 150 non-redundant serum proteins (excluding all immunoglobulin and albumin, a minimum of two peptide matches with acceptable Mascot score) in which 81 have not been identified previously in serum. Among them, numerous cellular proteins were identified to be specifically the skeletal muscle isoform, such as skeletal muscle fast twitch isoforms of troponin T, myosin alkali light chain 1, and sarcoplasmic/endoplasmic reticulum calcium ATPase. The detection of specific skeletal muscle protein isoforms in the serum from healthy individuals reflects the physiological turnover that occurs in skeletal muscle, which will have an impact on the ability to use generic "cellular" proteins as biomarkers without further characterization of the precise isoforms or post-translational modifications present. There has been a surge of interest in the proteomic analysis of plasma and serum in the search for clinically relevant biomarkers of disease. In biomarker discovery, it is necessary to maximize the observation of the plasma or serum proteome to detect proteins with low abundance. This can be achieved by optimization of protein separation methods as well as selective depletion of the proteins at high abundance such as immunoglobins (e.g. IgG) and albumin. There are a large number of proteomic technologies that separate either proteins or peptides prior to MS (1). A relative newcomer to the proteomic separation arena is the commercial instrument PF2D from Beckman Coulter that separates proteins in the first dimension using chromatographic focusing followed in line by reversed phase chromatography in the second dimension, thereby separating intact proteins based on pI and hydrophobicity. To date, assessment and optimization of either plasma or serum separation by the PF2D has not been undertaken. Generally LC proteomic methods have focused on separating complex mixtures of peptides obtained following digestion of the serum proteome (peptide LC, shotgun), whereas separation of proteins has been relegate...

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Section: Table II Optimization Of Chromatographic Focusing Conditionsmentioning

confidence: 99%

Multidimensional Liquid Chromatography Separation of Intact Proteins by Chromatographic Focusing and Reversed Phase of the Human Serum Proteome

Sheng

Chen

Eyk

2006

Molecular & Cellular Proteomics

102

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“…Trypsin was set as the enzyme for database search. All output results were combined together using the BuildingSummary software to delete keratins and the redundant data (27). For the analysis of BSA, the Sequest results were only filtered by the cross-correlation score (Xcorr).…”

Section: Methodsmentioning

confidence: 99%

Octadecylated Silica Monolith Capillary Column with Integrated Nanoelectrospray Ionization Emitter for Highly Efficient Proteome Analysis

Xie

Jiang

et al. 2006

Molecular & Cellular Proteomics

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An improved strategy for the preparation of octadecylated silica monolith capillary column with high homogeneity was proposed. Column performance was evaluated by nanoscale HPLC. The design for constructing an integrated nanoelectrospray emitter on the octadecylated silica monolith capillary column was first introduced. In comparison with the separated configuration where the emitter is connected to monolithic capillary column by the aid of a zero dead volume union, the integrated capillary column has the inherent advantage of the minimized extracolumn volume thus providing improved separation quality. The performance of the integrated monolithic capillary column was evaluated by separation of BSA tryptic digest, and peak capacity of 313 with a 30-cm column was obtained. The high separation performance allowed highly confident identification of 662 distinct proteins through assignment of 1933 unique peptides by analysis of tryptic digest of 0.5 g of Saccharomyces cerevisiae proteins. The higher separation efficiency by a 60-cm monolithic capillary column increased the proteome coverage with identification of 1323 proteins through assignment of 5501 unique peptides over 400-min gradient elution. Molecular & Cellular Proteomics 5:454 -461, 2006.The ultimate goal of proteomics is to study biological processes comprehensively by the systematic analysis of the proteins expressed in living systems (1). The basis for proteome analysis requires resolution of proteins followed by identification of the resolved proteins. Separation by twodimensional PAGE is the most widely used methodology in proteomic research (2, 3) as it combines two orthogonal separations to obtain efficient resolution of complex protein mixtures. However, two-dimensional PAGE has limitations such as difficulty to be automated and incompatibility with proteins of extreme pI values and molecular weight, low abundance proteins, and membrane-associated or -bound proteins (4 -6). Recently shotgun methodology based on nanoscale HPLC (nano-HPLC) 1 has emerged as an attractive alternative for proteome analysis because of its speed, ease of automation, and compatibility with mass spectrometry (7,8).Separation columns for nano-HPLC are usually fabricated by packing particulate beads with a controlled range of diameters and pore size. Particles of smaller diameters are preferably used to achieve a better efficiency, although they are hindered by the increase of backpressure. Recently the concept of monolithic column has been well established in separation technology (9 -13). The monolithic structure eliminates the interstitial voids in particulate columns, thus leading to fast mass transfer kinetics during separation. Moreover the enhanced permeability of monolithic rods results in a much lower backpressure, enabling the choice of longer columns to achieve increased separation performance (14,15). The pores present in the monolithic columns are key parameters affecting separation and permeability. As recommended by IUPAC, pores less than 2 nm in diameter are te...

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“…• Protein fractionation, depletion and/or enrichment (e.g., using ProteoMiner™ and multidimensional chromatography of proteins [35]) is needed to decrease the dynamic range at the protein level and, therefore, at the peptide level;…”

Section: Challengesmentioning

confidence: 99%

Peptide fractionation in proteomics approaches

Manadas

Mendes

English

et al. 2010

Expert Review of Proteomics

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Human Plasma Proteome Analysis by Multidimensional Chromatography Prefractionation and Linear Ion Trap Mass Spectrometry Identification

Cited by 76 publications

References 34 publications

Multidimensional Liquid Chromatography Separation of Intact Proteins by Chromatographic Focusing and Reversed Phase of the Human Serum Proteome

Multidimensional Liquid Chromatography Separation of Intact Proteins by Chromatographic Focusing and Reversed Phase of the Human Serum Proteome

Octadecylated Silica Monolith Capillary Column with Integrated Nanoelectrospray Ionization Emitter for Highly Efficient Proteome Analysis

Peptide fractionation in proteomics approaches

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