Efficient removal of detergents from proteins and peptides in a spin column format

Antharavally, Babu S.; Mallia, Krishna A; Rosenblatt, Michael; Salunkhe, Ashok M.; Rogers, John C.; Haney, Paul; Haghdoost, Navid

doi:10.1016/j.ab.2011.05.013

Cited by 45 publications

(46 citation statements)

References 15 publications

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“…It is also worth noting that, in this study, the detergent removal step included within the ISD protocol, carried out using spin columns, was performed on protein mixtures before trypsin digestion, while, according to several papers, similar columns were used to remove traces of detergent from peptide mixtures after digestion [24,46,47]. Previous papers have shown the successful application of an SDS depletion step on protein extracts before digestion, even leading to better results compared to FASP [48,49].…”

Section: Discussionmentioning

confidence: 99%

Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue

et al. 2014

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BackgroundThe growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research. Direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) or filter-aided sample preparation (FASP) are the most common workflows for shotgun analysis of FFPE samples, but a critical comparison of the different methods is currently lacking.Experimental designDT, FASP and ISD workflows were compared by subjecting to the same label-free quantitative approach three independent technical replicates of each method applied to FFPE liver tissue. Data were evaluated in terms of method reproducibility and protein/peptide distribution according to localization, MW, pI and hydrophobicity.ResultsDT showed lower reproducibility, good preservation of high-MW proteins, a general bias towards hydrophilic and acidic proteins, much lower keratin contamination, as well as higher abundance of non-tryptic peptides. Conversely, FASP and ISD proteomes were depleted in high-MW proteins and enriched in hydrophobic and membrane proteins; FASP provided higher identification yields, while ISD exhibited higher reproducibility.ConclusionsThese results highlight that diverse sample preparation strategies provide significantly different proteomic information, and present typical biases that should be taken into account when dealing with FFPE samples. When a sufficient amount of tissue is available, the complementary use of different methods is suggested to increase proteome coverage and depth.

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Section: Discussionmentioning

confidence: 99%

Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue

et al. 2014

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“…Moreover, the major drawback of SDS is its incompatibility with MS due to high ion suppression (42,43). To solve this problem, SDS removal using ion exchange chromatography (44), dialysis (45), ethyl acetate extraction (46), protein precipitation, use of a spin column removal kit (47), SDS precipitation (48), and filteraided sample preparation have been tested (24). Alternatively, detergents with properties similar to SDS but that are MScompatible have been used.…”

Section: Discussionmentioning

confidence: 99%

Discovery and Targeted Proteomics on Cutaneous Biopsies Infected by Borrelia to Investigate Lyme Disease*

Schnell

Bœuf

Westermann

et al. 2015

Molecular & Cellular Proteomics

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Lyme disease is the most important vector-borne disease in the Northern hemisphere and represents a major public health challenge with insufficient means of reliable diagnosis. Skin is rarely investigated in proteomics but constitutes in the case of Lyme disease the key interface where the pathogens can enter, persist, and multiply. Therefore, we investigated proteomics on skin samples to detect Borrelia proteins directly in cutaneous biopsies in a robust and specific way. We first set up a discovery gel prefractionation-LC-MS/MS approach on a murine model infected by Borrelia burgdorferi sensu stricto that allowed the identification of 25 Borrelia proteins among more than 1300 mouse proteins. Then we developed a targeted gel prefractionation-LC-selected reaction monitoring (SRM) assay to detect 9/33 Borrelia proteins/peptides in mouse skin tissue samples using heavy labeled synthetic peptides. We successfully transferred this assay from the mouse model to human skin biopsies (naturally infected by Borrelia), and we were able to detect two Borrelia proteins: OspC and flagellin. Considering the extreme variability of OspC, we developed an extended SRM assay to target a large set of variants. This assay afforded the detection of nine peptides belonging to either OspC or flagellin in human skin biopsies. We further shortened the sample preparation and showed that Borrelia is detectable in mouse and human skin biopsies by directly using a liquid digestion followed by LC-SRM analysis without any prefractionation. This study thus shows that a targeted SRM approach is a promising tool for the early direct diagnosis of Lyme disease with high sensitivity (<10 fmol of OspC/mg of human skin biopsy). Molecular & Cellular Proteomics

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“…However, this technology is incompatible with SDS since detergents interfere with antigen-antibody interactions by forming adducts with protein residues (7). Therefore, sample processing methods were adjusted to include an SDS exclusion step using commercially available, detergent-removing resin spin columns (Pierce).…”

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confidence: 99%

“…For large columns (25–100 μL sample volume), eluted protein was significantly less than loaded protein (P < 0.001) (N = 3) (Supplementary Figure S3B). Regardless of size, these spin columns function by providing a hydrophobic microenvironment in the resin cavity into which small nonpolar compounds, like SDS, infiltrate and subsequently form an inclusion complex (7). Non-specific adsorption or entrapment of protein is expected to be distributed evenly across protein types, resulting in a detergent-free protein lysate that is representative of the original sample proteome.…”

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confidence: 99%

Processing Fixed and Stored Adipose-Derived Stem Cells for Quantitative Protein Array Assays

Sadick

Darling

2017

BioTechniques

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Accurately characterizing cellular subpopulations is essential for elucidating the mechanisms underlying normal and pathological biology. Isolation of specific cell types can be accomplished by labeling unique, associated proteins with fluorescent antibodies. Cell fixation is commonly used to prepare these samples and allow for long-term storage, but this poses challenges for subsequent protein analysis. We previously established the FITSAR (Formaldehyde-fixed Intracellular Target-Sorted Antigen Retrieval) method in which protein can be isolated and characterized from fixed, enriched cell subpopulations. The current study improves on this method by allowing compatibility with highly sensitive, multiplex protein arrays and demonstrating applicability to long-term stored samples. Feasibility experiments demonstrated parallel detection of cell adhesion molecules using an enzyme-linked immunosorbent assay (ELISA) panel with human, adipose-derived stem cells stored for up to one month.

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Efficient removal of detergents from proteins and peptides in a spin column format

Cited by 45 publications

References 15 publications

Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue

Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue

Discovery and Targeted Proteomics on Cutaneous Biopsies Infected by Borrelia to Investigate Lyme Disease*

Processing Fixed and Stored Adipose-Derived Stem Cells for Quantitative Protein Array Assays

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