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Rosamonde E. Banks - One of the best experts on this subject based on the ideXlab platform.
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C-STrap Sample Preparation Method--In-Situ Cysteinyl Peptide Capture for Bottom-up Proteomics Analysis in the STrap Format.
PLOS ONE, 2015Co-Authors: Alexandre Zougman, Rosamonde E. BanksAbstract:Recently we introduced the concept of Suspension Trapping (STrap) for Bottom-up Proteomics sample processing that is based upon SDS-mediated protein extraction, swift detergent removal and rapid reactor-type protein digestion in a quartz depth filter trap. As the depth filter surface is made of silica, it is readily modifiable with various functional groups using the silane coupling chemistries. Thus, during the digest, peptides possessing specific features could be targeted for enrichment by the functionalized depth filter material while non-targeted peptides could be collected as an unbound distinct fraction after the digest. In the example presented here the quartz depth filter surface is functionalized with the pyridyldithiol group therefore enabling reversible in-situ capture of the cysteine-containing peptides generated during the STrap-based digest. The described C-STrap method retains all advantages of the original STrap methodology and provides robust foundation for the conception of the targeted in-situ peptide fractionation in the STrap format for Bottom-up Proteomics. The presented data support the method’s use in qualitative and semi-quantitative Proteomics experiments.
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suspension trapping strap sample preparation method for bottom up Proteomics analysis
Proteomics, 2014Co-Authors: Alexandre Zougman, Peter Selby, Rosamonde E. BanksAbstract:Despite recent developments in Bottom-up Proteomics, the need still exists in a fast, uncomplicated, and robust method for comprehensive sample processing especially when applied to low protein amounts. The suspension trapping method combines the advantage of efficient SDS-based protein extraction with rapid detergent removal, reactor-type protein digestion, and peptide cleanup. Proteins are solubilized in SDS. The sample is acidified and introduced into the suspension trapping tip incorporating the depth filter and hydrophobic compartments, filled with the neutral pH methanolic solution. The instantly formed fine protein suspension is trapped in the depth filter stack-this crucial step is aimed at separating the particulate matter in space. SDS and other contaminants are removed in the flow-through, and a protease is introduced. Following the digestion, the peptides are cleaned up using the tip's hydrophobic part. The methodology allows processing of protein loads down to the low microgram/submicrogram levels. The detergent removal takes about 5 min, whereas the tryptic proteolysis of a cellular lysate is complete in as little as 30 min. We have successfully utilized the method for analysis of cellular lysates, enriched membrane preparations, and immunoprecipitates. We expect that due to its robustness and simplicity, the method will become an essential Proteomics tool.
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Suspension trapping (STrap) sample preparation method for bottom‐up Proteomics analysis
Proteomics, 2014Co-Authors: Alexandre Zougman, Peter Selby, Rosamonde E. BanksAbstract:Despite recent developments in Bottom-up Proteomics, the need still exists in a fast, uncomplicated, and robust method for comprehensive sample processing especially when applied to low protein amounts. The suspension trapping method combines the advantage of efficient SDS-based protein extraction with rapid detergent removal, reactor-type protein digestion, and peptide cleanup. Proteins are solubilized in SDS. The sample is acidified and introduced into the suspension trapping tip incorporating the depth filter and hydrophobic compartments, filled with the neutral pH methanolic solution. The instantly formed fine protein suspension is trapped in the depth filter stack-this crucial step is aimed at separating the particulate matter in space. SDS and other contaminants are removed in the flow-through, and a protease is introduced. Following the digestion, the peptides are cleaned up using the tip's hydrophobic part. The methodology allows processing of protein loads down to the low microgram/submicrogram levels. The detergent removal takes about 5 min, whereas the tryptic proteolysis of a cellular lysate is complete in as little as 30 min. We have successfully utilized the method for analysis of cellular lysates, enriched membrane preparations, and immunoprecipitates. We expect that due to its robustness and simplicity, the method will become an essential Proteomics tool.
Ruedi Aebersold - One of the best experts on this subject based on the ideXlab platform.
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Systematic detection of functional proteoform groups from Bottom-up proteomic datasets
2020Co-Authors: I. Bludau, Max Frank, Ben C. Collins, C. Doerig, Y. Cai, Moritz Heusel, George Rosenberger, P. Picotti, H. Roest, Ruedi AebersoldAbstract:The cellular proteome, the ensemble of proteins derived from a genome, catalyzes and controls thousands of biochemical functions that are the basis of living cells. Whereas the protein coding regions of the genome of the human and many other species are well known, the complexity and composition of proteomes largely remains to be explored. This task is challenging because mechanisms including alternative splicing and post-translational modifications generally give rise to multiple distinct, but related proteins - proteoforms - per coding gene that expand the functional capacity of a cell. Bottom-up Proteomics is a mass spectrometric method that infers the identity and quantity of proteins from the measurement of peptides derived from these proteins by proteolytic digestion. Whereas Bottom-up Proteomics has become the method of choice for the detection of translation products from essentially any gene, the inherent missing link between measured peptides and their parental proteins has so far precluded the systematic assessment of proteoforms and thus limited the resolution of proteome maps. Here we present a novel, data-driven strategy to assign peptides to unique functional proteoform groups based on peptide correlation patterns across large Bottom-up proteomic datasets. Our strategy does not fully characterize specific proteoforms, as is achievable in top-down approaches. Rather, it clusters peptides into functional proteoform groups that are directly linked to the biological context of the study. This allows the detection of tens to hundreds of proteoform groups in an untargeted fashion from Bottom-up Proteomics experiments. We applied the strategy to two types of Bottom-up proteomic datasets. The first is a protein complex co-fractionation dataset where native complexes across two different cell cycle stages were resolved and analyzed. Here, our approach enabled the systematic detection and evaluation of assembly specific proteoforms at an unprecedented scale. The second is a protein abundance vs. sample data matrix typical for Bottom-up cohort studies consisting of tissue samples from the mouse BXD genetic reference panel. In either data type the method detected state-specific proteoform groups that could be linked to distinct molecular mechanisms including proteolytic cleavage, alternative splicing and phosphorylation. We envision that the presented approach lays the foundation for a systematic assessment of proteoforms and their functional implications directly from Bottom-up proteomic datasets.
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Parallel accumulation – serial fragmentation combined with data-independent acquisition (diaPASEF): Bottom-up Proteomics with near optimal ion usage
2019Co-Authors: Florian Meier, Ruedi Aebersold, Andreas-david Brunner, Max Frank, Eugenia Voytik, Stephanie Kaspar-schoenefeld, Markus Lubeck, Oliver Raether, Ben C. CollinsAbstract:ABSTRACT Bottom-up Proteomics produces complex peptide populations that are identified and quantified at the precursor or fragment ion level. Data dependent acquisition methods sequentially isolate and fragment particular precursors, whereas data independent acquisition (DIA) modes isolate and concurrently fragment populations of different precursors by cycling deterministically through segments of a predefined precursor m/z range. Although the selection windows of DIA collectively cover the entire mass range of interest, only a few percent of the ion current are sampled due to the consecutive selection of acquisition windows. Making use of the correlation of molecular weight and ion mobility in a trapped ion mobility device (timsTOF Pro), we here devise a novel scan mode that samples up to 100% of the peptide precursor ion current. We analyze the acquired data by extending established targeted data extraction workflow for the analysis of DIA data by the additional ion mobility dimension, providing additional specificity in the precursor identification. Data acquired from simple protein mixtures verify the expected data completeness and data in single runs of a whole proteome digest demonstrate deep proteome coverage and a very high degree of reproducibility and quantitative accuracy, even from 10 ng sample amounts.
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Mass Spectrometry Applied to Bottom-up Proteomics: Entering the High-Throughput Era for Hypothesis Testing.
Annual review of analytical chemistry (Palo Alto Calif.), 2016Co-Authors: Ludovic C. Gillet, Alexander Leitner, Ruedi AebersoldAbstract:Proteins constitute a key class of molecular components that perform essential biochemical reactions in living cells. Whether the aim is to extensively characterize a given protein or to perform high-throughput qualitative and quantitative analysis of the proteome content of a sample, liquid chromatography coupled to tandem mass spectrometry has become the technology of choice. In this review, we summarize the current state of mass spectrometry applied to Bottom-up Proteomics, the approach that focuses on analyzing peptides obtained from proteolytic digestion of proteins. With the recent advances in instrumentation and methodology, we show that the field is moving away from providing qualitative identification of long lists of proteins to delivering highly consistent and accurate quantification values for large numbers of proteins across large numbers of samples. We believe that this shift will have a profound impact for the field of Proteomics and life science research in general.
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peptide centric proteome analysis an alternative strategy for the analysis of tandem mass spectrometry data
Molecular & Cellular Proteomics, 2015Co-Authors: Ying S Ting, Samuel H. Payne, Lukas Käll, Richard D Smith, Ruedi Aebersold, Jarrett D Egertson, Brendan Maclean, William Stafford Noble, Michael J. MaccossAbstract:In mass spectrometry-based Bottom-up Proteomics, data-independent acquisition is an emerging technique because of its comprehensive and unbiased sampling of precursor ions. However, current data-in ...
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DIGESTIF: a universal quality standard for the control of Bottom-up Proteomics experiments.
Journal of proteome research, 2014Co-Authors: Dorothée Lebert, Mathilde Louwagie, Sandra Goetze, Guillaume Picard, Reto Ossola, Caroline Duquesne, Konrad Basler, Myriam Ferro, Oliver Rinner, Ruedi AebersoldAbstract:In Bottom-up mass spectrometry-based Proteomics analyses, variability at any step of the process, particularly during sample proteolysis, directly affects the sensitivity, accuracy, and precision of peptide detection and quantification. Currently, no generic internal standards are available to control the quality of sample processing steps. This makes it difficult to assess the comparability of MS proteomic data obtained under different experimental conditions. Here, we describe the design, synthesis, and validation of a universal protein standard, called DIGESTIF, that can be added to any biological sample. The DIGESTIF standard consists of a soluble recombinant protein scaffold to which a set of 11 artificial peptides (iRT peptides) with good ionization properties has been incorporated. In the protein scaffold, the amino acids flanking iRT peptide cleavage sites were selected either to favor or hinder protease cleavage. After sample processing, the retention time and relative intensity pattern of the re...
Liangliang Sun - One of the best experts on this subject based on the ideXlab platform.
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Microscale Reversed-Phase Liquid Chromatography/Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Deep and Highly Sensitive Bottom–Up Proteomics: Identification of 7500 Proteins with Five Micrograms of an MCF7 Proteome Digest
Analytical chemistry, 2018Co-Authors: Zhichang Yang, Xiaojing Shen, Daoyang Chen, Liangliang SunAbstract:Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has been well recognized for bottom–up Proteomics. It has approached 4000–8000 protein identifications (IDs) from a human cell line, mouse brains, or Xenopus embryos via coupling with liquid chromatography (LC) prefractionation. However, at least 500 μg of complex proteome digests were required for the LC/CZE-MS/MS studies. This requirement of a large amount of initial peptide material impedes the application of CZE-MS/MS for deep bottom–up Proteomics of mass-limited samples. In this work, we coupled microscale reversed-phase LC (μRPLC)-based peptide prefractionation to dynamic pH-junction-based CZE-MS/MS for deep bottom–up Proteomics of the MCF7 breast cancer cell proteome starting with only 5 μg of peptides. The dynamic pH-junction-based CZE enabled a 500 nL sample injection from as low as a 1.5 μL peptide sample, using up to 33% of the available peptide material for an analysis. Two kinds of μRPLC prefractionation were investigated, C1...
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strong cation exchange reversed phase liquid chromatography capillary zone electrophoresis tandem mass spectrometry platform with high peak capacity for deep bottom up Proteomics
Analytica Chimica Acta, 2018Co-Authors: Daoyang Chen, Xiaojing Shen, Liangliang SunAbstract:Abstract Two-dimensional (2D) liquid chromatography (LC)-tandem mass spectrometry (MS/MS) are typically employed for deep Bottom-up Proteomics, and the state-of-the-art 2D-LC-MS/MS has approached over 8000 protein identifications (IDs) from mammalian cell lines or tissues in 1–3 days of mass spectrometer time. Capillary zone electrophoresis (CZE)-MS/MS has been suggested as an alternative to LC-MS/MS for Bottom-up Proteomics. CZE-MS/MS and LC-MS/MS are complementary in protein/peptide ID from complex proteome digests because CZE and LC are orthogonal for peptide separation. In addition, the migration time of peptides from CZE-MS can be predicted accurately, which is invaluable for evaluating the confidence of peptide ID from the database search and even guiding the database search. However, the number of protein IDs from complex proteomes using CZE-MS/MS is still much lower than the state of the art using 2D-LC-MS/MS. In this work, for the first time, we established a strong cation exchange (SCX)-reversed phase LC (RPLC)-CZE-MS/MS platform for deep Bottom-up Proteomics. The platform identified around 8200 protein groups and 65,000 unique peptides from a mouse brain proteome digest in 70 h. The data represents the largest Bottom-up Proteomics dataset using CZE-MS/MS and provides a valuable resource for further improving the tool for prediction of peptide migration time in CZE. The peak capacity of the orthogonal SCX-RPLC-CZE platform was estimated to be around 7000. SCX-RPLC-CZE-MS/MS produced comparable numbers of protein and peptide IDs with 2D-LC-MS/MS (8200 vs. 8900 protein groups, 65,000 vs. 70,000 unique peptides) from the mouse brain proteome digest using comparable instrument time. This is the first time that CZE-MS/MS showed its capability to approach comparable performance to the state-of-the-art 2D-LC-MS/MS for deep proteomic sequencing. SCX-RPLC-CZE-MS/MS and 2D-LC-MS/MS showed good complementarity in protein and peptide IDs and combining those two methods improved the number of protein group and unique peptide IDs by nearly 10% and over 40%, respectively, compared with 2D-LC-MS/MS alone.
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Systematic Evaluation of Immobilized Trypsin-Based Fast Protein Digestion for Deep and High-Throughput Bottom-up Proteomics.
Proteomics, 2018Co-Authors: Xiaojing Shen, Liangliang SunAbstract:Immobilized trypsin (IM) has been recognized as an alternative to free trypsin (FT) for accelerating protein digestion 30 years ago. However, some questions of IM still need to be answered. How does the solid matrix of IM influence its preference for protein cleavage and how well can IM perform for deep Bottom-up Proteomics compared to FT? By analyzing Escherichia coli proteome samples digested with amine or carboxyl functionalized magnetic bead-based IM (IM-N or IM-C) or FT, it is observed that IM-N with the nearly neutral solid matrix, IM-C with the negatively charged solid matrix, and FT have similar cleavage preference considering the microenvironment surrounding the cleavage sites. IM-N (15 min) and FT (12 h) both approach 9000 protein identifications (IDs) from a mouse brain proteome. Compared to FT, IM-N has no bias in the digestion of proteins that are involved in various biological processes, are located in different components of cells, have diverse functions, and are expressed in varying abundance. A high-throughput Bottom-up Proteomics workflow comprising IM-N-based rapid protein cleavage and fast CZE-MS/MS enables the completion of protein sample preparation, CZE-MS/MS analysis, and data analysis in only 3 h, resulting in 1000 protein IDs from the mouse brain proteome.
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capillary zone electrophoresis mass spectrometry with microliter scale loading capacity 140 min separation window and high peak capacity for bottom up Proteomics
Analyst, 2017Co-Authors: Daoyang Chen, Xiaojing Shen, Liangliang SunAbstract:Better peptide separation is required for Bottom-up Proteomics for further improving the proteome coverage. The two-dimensional liquid chromatography (2D-LC) systems only explore differences among peptides in their hydrophobicity (reversed-phase, RP) and charge (strong cation/anion exchange, SCX/SAX). Alternative separation techniques with different separation mechanisms are required to further improve the separation. Capillary zone electrophoresis (CZE) is an attractive alternative because it has high efficiency for separation of biomolecules and it separates analytes based on their size-to-charge ratios, complementary with LC. However, the low loading capacity and narrow separation window of CZE limit its wide application for large-scale Proteomics. In this manuscript, we present an automated CZE–mass spectrometry (MS) system for solving those issues. The CZE–MS system can approach at least half-a-microliter loading capacity with good robustness and reproducibility, can routinely use over 12% of the available sample in the sample vial for analysis, and can generate a 140 min separation window and high peak capacity (∼380) for complex proteome analysis. The results represent the highest peak capacity and the widest separation window of CZE for peptide separation with a microliter-scale loading capacity. It is the first time that CZE–MS approaches both the microliter-scale loading capacity and over 2-hour separation window for analysis of complex samples. The automated CZE–MS system dramatically reduces the gap between CZE–MS and RPLC–MS in terms of loading capacity, separation window and peak capacity. It truly opens the door for large-scale Bottom-up Proteomics using CZE–MS.
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Capillary zone electrophoresis–mass spectrometry with microliter-scale loading capacity, 140 min separation window and high peak capacity for Bottom-up Proteomics
The Analyst, 2017Co-Authors: Daoyang Chen, Xiaojing Shen, Liangliang SunAbstract:Better peptide separation is required for Bottom-up Proteomics for further improving the proteome coverage. The two-dimensional liquid chromatography (2D-LC) systems only explore differences among peptides in their hydrophobicity (reversed-phase, RP) and charge (strong cation/anion exchange, SCX/SAX). Alternative separation techniques with different separation mechanisms are required to further improve the separation. Capillary zone electrophoresis (CZE) is an attractive alternative because it has high efficiency for separation of biomolecules and it separates analytes based on their size-to-charge ratios, complementary with LC. However, the low loading capacity and narrow separation window of CZE limit its wide application for large-scale Proteomics. In this manuscript, we present an automated CZE–mass spectrometry (MS) system for solving those issues. The CZE–MS system can approach at least half-a-microliter loading capacity with good robustness and reproducibility, can routinely use over 12% of the available sample in the sample vial for analysis, and can generate a 140 min separation window and high peak capacity (∼380) for complex proteome analysis. The results represent the highest peak capacity and the widest separation window of CZE for peptide separation with a microliter-scale loading capacity. It is the first time that CZE–MS approaches both the microliter-scale loading capacity and over 2-hour separation window for analysis of complex samples. The automated CZE–MS system dramatically reduces the gap between CZE–MS and RPLC–MS in terms of loading capacity, separation window and peak capacity. It truly opens the door for large-scale Bottom-up Proteomics using CZE–MS.
Norman J. Dovichi - One of the best experts on this subject based on the ideXlab platform.
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Capillary zone electrophoresis-mass spectrometry for Bottom-up Proteomics
TrAC Trends in Analytical Chemistry, 2018Co-Authors: Zhenbin Zhang, Norman J. DovichiAbstract:Abstract Bottom-up Proteomics characterizes proteins by analysis of peptides generated through proteolysis. Bottom-up analysis of a complex proteome inevitably generates tens of thousands of peptides, and the analysis of these peptides is a serious challenge. Capillary zone electrophoresis (CZE) generates separations that are orthogonal to reversed phase liquid chromatography, which has led to consideration of CZE as an alternative separation technology in proteomic analysis. The steady improvement in mass spectrometer (MS) technology coupled with improvements in capillary coatings and the development of robust CZE-MS interfaces have contributed to the rapid advancement of CZE's identification performance in Bottom-up Proteomics analysis. In this review, we focus on recent advances of CZE-MS based Bottom-up Proteomics, including optimization of CZE and MS conditions, and the application of CZE-MS in phosphoProteomics, glycoProteomics, clinical diagnosis, host cell protein analysis, ultrasensitive Proteomics, and quantitative Proteomics. Finally, we outline future opportunities and challenges in this field.
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Optimization of mass spectrometric parameters improve the identification performance of capillary zone electrophoresis for single-shot Bottom-up Proteomics analysis.
Analytica chimica acta, 2017Co-Authors: Zhenbin Zhang, Norman J. DovichiAbstract:Abstract The effects of MS1 injection time, MS2 injection time, dynamic exclusion time, intensity threshold, and isolation width were investigated on the numbers of peptide and protein identifications for single-shot Bottom-up Proteomics analysis using CZE-MS/MS analysis of a Xenopus laevis tryptic digest. An electrokinetically pumped nanospray interface was used to couple a linear-polyacrylamide coated capillary to a Q Exactive HF mass spectrometer. A sensitive method that used a 1.4 Th isolation width, 60,000 MS2 resolution, 110 ms MS2 injection time, and a top 7 fragmentation produced the largest number of identifications when the CZE loading amount was less than 100 ng. A programmable autogain control method (pAGC) that used a 1.4 Th isolation width, 15,000 MS2 resolution, 110 ms MS2 injection time, and top 10 fragmentation produced the largest number of identifications for CZE loading amounts greater than 100 ng; 7218 unique peptides and 1653 protein groups were identified from 200 ng by using the pAGC method. The effect of mass spectrometer conditions on the performance of UPLC-MS/MS was also investigated. A fast method that used a 1.4 Th isolation width, 30,000 MS2 resolution, 45 ms MS2 injection time, and top 12 fragmentation produced the largest number of identifications for 200 ng UPLC loading amount (6025 unique peptides and 1501 protein groups). This is the first report where the identification number for CZE surpasses that of the UPLC at the 200 ng loading level. However, more peptides (11476) and protein groups (2378) were identified by using UPLC-MS/MS when the sample loading amount was increased to 2 μg with the fast method. To exploit the fast scan speed of the Q-Exactive HF mass spectrometer, higher sample loading amounts are required for single-shot Bottom-up Proteomics analysis using CZE-MS/MS.
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Multisegment injections improve peptide identification rates in capillary zone electrophoresis-based Bottom-up Proteomics.
Journal of chromatography. A, 2017Co-Authors: Danielle A. Boley, Zhenbin Zhang, Norman J. DovichiAbstract:While capillary zone electrophoresis (CZE) provides dramatically improved numbers of peptide identifications compared with reversed-phase chromatography for Bottom-up Proteomics of mass limited samples, CZE inevitably produces lower numbers of peptide identifications than RPLC for larger samples. One reason for this poorer performance is the dead time between injection of samples and subsequent appearance of the fastest moving component. This dead time is typically 25% of the separation window in CZE, but is only 5% of the separation window in gradient elution RPLC. This dead time can be eliminated in CZE by use of a multisegment injection mode where a series of samples is analyzed by injecting each sample while the preceding sample is still being separated. In this paper, we demonstrate that capillary zone electrophoresis employing sequential injections can produce a doubling in peptide identification rate with no degradation in separation efficiency.
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bottom up Proteomics analysis of the secretome of murine islets of langerhans in elevated glucose levels
Analyst, 2017Co-Authors: Andrew Schmudlach, Jeremy Felton, Robert T Kennedy, Norman J. DovichiAbstract:Glucotoxicity is a causative agent of type-2 diabetes, where high glucose levels damage the islets of Langerhans resulting in oxidative damage and endoplasmic reticulum stress. We evaluated the secretomes of healthy CD-1 murine islets. Three experimental conditions were investigated in biological triplicate: a control incubated with 11 mM glucose, 1-day incubation with 25 mM glucose, and 2-day incubation with 25 mM glucose. An SDS-based, filter-aided sample preparation protocol was used to prepare secretomes for analysis. A total of 428 protein groups were identified across the nine samples. Each condition generated between 328–349 protein IDs and intracondition protein overlap was between 66–90% for the biological triplicates. 232 protein groups were identified in all three conditions with 184 quantified at least once in each condition. Significant expression changes were observed for proteins associated with the unfolded protein response, such as proteases, chaperones, and elongation factors, as well as proteins associated with peptide hormone processing and small molecule metabolism.
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Bottom-up Proteomics of Escherichia coli using dynamic pH junction preconcentration and capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry.
Analytical chemistry, 2014Co-Authors: Guijie Zhu, Liangliang Sun, Xiaojing Yan, Norman J. DovichiAbstract:We report the use of the dynamic pH junction based capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) for Bottom-up Proteomics with an electrokinetically pumped sheath-flow nanospray capillary electrophoresis-mass spectrometry (CE-MS) interface and both LTQ-XL and LTQ-Orbitrap-Velos mass spectrometers. Conventional injection of 20 nL of a 1 mg/mL BSA digest identified 37 peptides and produced 66% sequence coverage. In contrast, pH junction injection of 130 nL (or larger) of a 0.05 mg/mL BSA digest identified 40 peptides and produced 70% coverage using a pH 6.5 sample buffer and the LTQ. A 20 nL conventional injection of a 1 mg/mL Escherichia coli digest identified 508 peptides and 199 proteins with the LTQ. A 400 nL pH junction injection of a 0.1 mg/mL E. coli digest identified 527 peptides and 179 proteins with the LTQ. Triplicate technical replicates of a 0.01 mg/mL sample with 400-nL injection volume using a pH junction identified 288 ± 9 peptides and 121 ± 5 proteins with the LTQ. There was outstanding concordance in migration time between the pH junction and normal injection. The pH junction produced narrower peaks and significant concentration for all but the most acidic components in the sample. Compared with the conventional stacking method, the pH junction method can generate comparable performance for small injection volume (20 nL) and significantly better concentration performance for a large injection volume (200 nL). We also applied the pH junction to three intact standard proteins and observed a >10× increase in peak intensity compared to conventional injection.
Alexandre Zougman - One of the best experts on this subject based on the ideXlab platform.
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C-STrap Sample Preparation Method--In-Situ Cysteinyl Peptide Capture for Bottom-up Proteomics Analysis in the STrap Format.
PLOS ONE, 2015Co-Authors: Alexandre Zougman, Rosamonde E. BanksAbstract:Recently we introduced the concept of Suspension Trapping (STrap) for Bottom-up Proteomics sample processing that is based upon SDS-mediated protein extraction, swift detergent removal and rapid reactor-type protein digestion in a quartz depth filter trap. As the depth filter surface is made of silica, it is readily modifiable with various functional groups using the silane coupling chemistries. Thus, during the digest, peptides possessing specific features could be targeted for enrichment by the functionalized depth filter material while non-targeted peptides could be collected as an unbound distinct fraction after the digest. In the example presented here the quartz depth filter surface is functionalized with the pyridyldithiol group therefore enabling reversible in-situ capture of the cysteine-containing peptides generated during the STrap-based digest. The described C-STrap method retains all advantages of the original STrap methodology and provides robust foundation for the conception of the targeted in-situ peptide fractionation in the STrap format for Bottom-up Proteomics. The presented data support the method’s use in qualitative and semi-quantitative Proteomics experiments.
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suspension trapping strap sample preparation method for bottom up Proteomics analysis
Proteomics, 2014Co-Authors: Alexandre Zougman, Peter Selby, Rosamonde E. BanksAbstract:Despite recent developments in Bottom-up Proteomics, the need still exists in a fast, uncomplicated, and robust method for comprehensive sample processing especially when applied to low protein amounts. The suspension trapping method combines the advantage of efficient SDS-based protein extraction with rapid detergent removal, reactor-type protein digestion, and peptide cleanup. Proteins are solubilized in SDS. The sample is acidified and introduced into the suspension trapping tip incorporating the depth filter and hydrophobic compartments, filled with the neutral pH methanolic solution. The instantly formed fine protein suspension is trapped in the depth filter stack-this crucial step is aimed at separating the particulate matter in space. SDS and other contaminants are removed in the flow-through, and a protease is introduced. Following the digestion, the peptides are cleaned up using the tip's hydrophobic part. The methodology allows processing of protein loads down to the low microgram/submicrogram levels. The detergent removal takes about 5 min, whereas the tryptic proteolysis of a cellular lysate is complete in as little as 30 min. We have successfully utilized the method for analysis of cellular lysates, enriched membrane preparations, and immunoprecipitates. We expect that due to its robustness and simplicity, the method will become an essential Proteomics tool.
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Suspension trapping (STrap) sample preparation method for bottom‐up Proteomics analysis
Proteomics, 2014Co-Authors: Alexandre Zougman, Peter Selby, Rosamonde E. BanksAbstract:Despite recent developments in Bottom-up Proteomics, the need still exists in a fast, uncomplicated, and robust method for comprehensive sample processing especially when applied to low protein amounts. The suspension trapping method combines the advantage of efficient SDS-based protein extraction with rapid detergent removal, reactor-type protein digestion, and peptide cleanup. Proteins are solubilized in SDS. The sample is acidified and introduced into the suspension trapping tip incorporating the depth filter and hydrophobic compartments, filled with the neutral pH methanolic solution. The instantly formed fine protein suspension is trapped in the depth filter stack-this crucial step is aimed at separating the particulate matter in space. SDS and other contaminants are removed in the flow-through, and a protease is introduced. Following the digestion, the peptides are cleaned up using the tip's hydrophobic part. The methodology allows processing of protein loads down to the low microgram/submicrogram levels. The detergent removal takes about 5 min, whereas the tryptic proteolysis of a cellular lysate is complete in as little as 30 min. We have successfully utilized the method for analysis of cellular lysates, enriched membrane preparations, and immunoprecipitates. We expect that due to its robustness and simplicity, the method will become an essential Proteomics tool.