Top-Down and Bottom-Up Proteomics of SDS-Containing Solutions Following Mass-Based Separation

Botelho, Diane; Wall, Mark J.; Vieira, Douglas B.; Fitzsimmons, Shayla; Liu, Fang; Doucette, Alan A.

doi:10.1021/pr900949p

Cited by 151 publications

(195 citation statements)

References 30 publications

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“…However, this criterion presents a major challenge for plant proteomic research using electrospray mass spectrometry, as detergent-containing solutions can impede enzymatic digestion and cause significant analyte suppression (9). Therefore, most plant proteomic studies using the "MudPIT" strategy apply mechanical disruption in conjunction with a detergentfree preparation method (10).…”

mentioning

confidence: 99%

Putting the Pieces Together: High-performance LC-MS/MS Provides Network-, Pathway-, and Protein-level Perspectives in Populus

Abraham

Giannone

Adams

et al. 2013

Molecular & Cellular Proteomics

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High-performance mass spectrometry (MS)-based proteomics enabled the construction of a detailed proteome atlas for Populus, a woody perennial plant model organism. Optimization of experimental procedures and implementation of current state-of-the-art instrumentation afforded the most detailed look into the predicted proteome space of Populus, offering varying proteome perspectives: (1) network-wide, (2) pathway-specific, and (3) protein-level viewpoints. Together, enhanced protein retrieval through a detergent-based lysis approach and maximized peptide sampling via the dual-pressure linear ion trap mass spectrometer (LTQ Velos), have resulted in the identification of 63,056 tryptic peptides. The technological advancements, specifically spectral-acquisition and sequencing speed, afforded the deepest look into the Populus proteome, with peptide abundances spanning 6 orders of magnitude and mapping to ϳ25% of the predicted proteome space. In total, tryptic peptides mapped to 11,689 protein assignments across four organ-types: mature (fully expanded, leaf plastichronic index (LPI) 10 -12) leaf, young (juvenile, LPI 4 -6) leaf, root, and stem. To resolve protein ambiguity, identified proteins were grouped by sequence similarity (> 90%), thereby reducing the protein assignments into 7538 protein groups. In addition, this large-scale data set features the first systemswide survey of protein expression across different Populus organs. As a demonstration of the precision and comprehensiveness of the semiquantitative analysis, we were able to contrast two stages of leaf development, mature versus young leaf. Statistical comparison through ANOVA analysis revealed 1432 protein groups that exhibited statistically significant (p < 0.01) differences in protein abundance. Experimental validation of the metabolic circuitry expected in mature leaf (characterized by photosynthesis and carbon fixation) compared with young leaf (characterized by rapid growth and moderate photosynthetic activities) strongly testifies to the credibility of the approach. Mass spectrometry (MS)-based proteomics has experienced tremendous growth in recent years, leading to the establishment of numerous protocols, platforms, and workflows for the characterization of protein expression at the genome level (1). Although these advancements have facilitated comprehensive proteomic investigations of simple bacterial isolates and microbial communities, the application of MS-based proteomics for plants and other higher eukaryotes remains underdeveloped. Recently, large-scale proteomic studies have been directed at characterization of Populus, a woody perennial model organism. With the recent release and subsequent curation of the P. trichocarpa genome (2), these large-scale MS-based proteomic investigations offer the potential to introduce new biological insights into woody perennial plant biology (3,4,5). For example, we have recently demonstrated the ability to measure ϳ17% of the Populus proteome by coupling multidimensional liquid chromatography (MudPIT) 1 w...

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mentioning

confidence: 99%

Putting the Pieces Together: High-performance LC-MS/MS Provides Network-, Pathway-, and Protein-level Perspectives in Populus

Abraham

Giannone

Adams

et al. 2013

Molecular & Cellular Proteomics

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“…Of the solution-based methods that can be used to clean up protein samples, protein precipitation with organic solvents, especially with cold acetone, has attracted special attention and found some applications (Puchades et al, 1999;Botelho et al, 2010). The method operates simply and can reduce the concentration of small-molecular-weight interfering substances in protein samples, thus reducing the effects of these substances on subsequent digestion and mass spectrometric analysis.…”

Section: Entirely Solution-based Sample Preparationmentioning

confidence: 99%

Sample preparation for the analysis of membrane proteomes by mass spectrometry

Wang

Liang

2012

Protein Cell

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The low abundance and highly hydrophobic nature of most membrane proteins make their analysis more difficult than that for common soluble proteins. Successful membrane protein identification is largely dependent on the sample preparation including the enrichment and dissolution of the membrane proteins. A series of conventional and newly developed methods has been applied to the enrichment of low-abundance membrane proteins at membrane and/or protein levels and to the dissolution of hydrophobic membrane proteins. However, all the existing methods have inherent advantages and limitations. Up to now, there has been no unique method that can universally be employed to solve all the problems and more efforts are needed in improving sample preparation for the analysis of membrane proteomes.

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“…Accuracy and reliability of bottom-up mass spectrometry (MS)-based proteomics analyses are undoubtedly dependent on the quality of prior sample preparation, which remains very challenging 3 . In fact, sodium dodecyl sulfate (SDS) is a powerful anionic detergent and one of the most widely used reagents for solubilisation and denaturation of proteins 4 . By binding amino acids through hydrophobic and ionic interactions, SDS alters proteins' spatial conformational structure and inhibits proteases activity, thus enabling long-term conservation of structurally preserved proteins 5 .…”

Section: Introductionmentioning

confidence: 99%

“…By binding amino acids through hydrophobic and ionic interactions, SDS alters proteins' spatial conformational structure and inhibits proteases activity, thus enabling long-term conservation of structurally preserved proteins 5 . In spite of all its advantages, SDS significantly suppresses analyte ion signals in electrospray ionization (ESI)-MS, alters the chromatographic separation of peptides during liquid chromatography (LC) 4,6 , and strongly inhibits trypsin activity 7 , even at very low concentrations, which considerably limits protein identification. Samples therefore require to be completely depleted of SDS prior to digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.…”

Section: Introductionmentioning

confidence: 99%

Amicon-adapted enhanced FASP: an in-solution digestion-based alternative sample preparation method to FASP

et al. 2015

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Sample preparation is a crucial step for liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics. Sodium dodecyl sulfate (SDS) is a powerful denaturing detergent that allows for long-term preservation of protein integrity. However, as it inhibits trypsin and interferes with LC-MS/MS analyses, it must be removed from samples prior to these experiments. The Filter-Aided Sample Preparation (FASP) method is actually one of the preferred and simplest methods for such purpose. Nonetheless, there exist great disparities in the quality of outcomes when comparing FASP to other protocols depending on the authors, and recent reports have pointed to concerns regarding its depth of proteome coverage. To address these issues, we propose an Amicon-adapted in-solution-based enhanced FASP (eFASP) approach that relies on current best practices in comprehensive proteomics sample preparation. Human megakaryoblastic leukaemia cancer cells' protein extracts were treated in parallel with both Amicon-adapted eFASP and FASP, quantified for remaining SDS and then analyzed with a 1-hr gradient LC-MS/MS run. The Amicon-adapted eFASP utilizes a passivated low molecular weight cut-off Amicon filter, and incorporates a cleaning step with a high-content deoxycholate buffer and a 'one-step-two-enzymes' trypsin/Lys-C in-solution digestion. Amicon-adapted eFASP was found more reproducible and deepened proteome coverage, especially for membrane proteins. As compared to FASP, Amicon-adapted eFASP removed much of SDS from high-protein samples and reached a notable depth of proteome coverage with nearly 1,700 proteins identified in a 1 hr LC-MS/MS single-run analysis without prior fractionation. Amicon-adapted eFASP can therefore be regarded as a simple and reliable sample preparation approach for comprehensive proteomics.

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Top-Down and Bottom-Up Proteomics of SDS-Containing Solutions Following Mass-Based Separation

Cited by 151 publications

References 30 publications

Putting the Pieces Together: High-performance LC-MS/MS Provides Network-, Pathway-, and Protein-level Perspectives in Populus

Putting the Pieces Together: High-performance LC-MS/MS Provides Network-, Pathway-, and Protein-level Perspectives in Populus

Sample preparation for the analysis of membrane proteomes by mass spectrometry

Amicon-adapted enhanced FASP: an in-solution digestion-based alternative sample preparation method to FASP

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