Sample preparation protocol for bottom-up proteomic analysis of the secretome of the islets of Langerhans

Schmudlach, Andrew; Felton, Jeremy; Cipolla, Cynthia M.; Sun, Liangliang; Kennedy, Robert T.; Dovic̀hi, Norman J.

doi:10.1039/c5an02265g

Cited by 18 publications

(17 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…24 We have now extended that earlier study to consider the effect of high glucose treatments on the secretome. The CD-1 mouse islets generate highly reproducible secretomes when analyzed by the FASP protocol.…”

Section: Discussionmentioning

confidence: 85%

“…24 Pancreata were injected with 2-3 mL of collagenase and placed in 1 mL of G solution. Islets were isolated from the interface of a Histopasque-1077 and HBSS gradient separation.…”

Section: Methodsmentioning

confidence: 99%

“…24 Insulin and other peptides are depleted using a 30-kDa molecular-weight cutoff filter. This depletion allows a deeper analysis of higher molecular weight components of the secretome by removing highly abundant insulin and C-peptide.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Bottom-up proteomics analysis of the secretome of murine islets of Langerhans in elevated glucose levels

Schmudlach

Felton

Kennedy

et al. 2017

Analyst

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 85%

“…24 Pancreata were injected with 2-3 mL of collagenase and placed in 1 mL of G solution. Islets were isolated from the interface of a Histopasque-1077 and HBSS gradient separation.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Bottom-up proteomics analysis of the secretome of murine islets of Langerhans in elevated glucose levels

Schmudlach

Felton

Kennedy

et al. 2017

Analyst

Self Cite

View full text Add to dashboard Cite

show abstract

“…We have recently compared several sample preparation protocols for generation of the secretome from murine islets of Langerhans [23]. That sample is much simpler than Xenopus embryos, and generated far fewer protein identifications.…”

Section: Resultsmentioning

confidence: 99%

Optimization and comparison of bottom-up proteomic sample preparation for early-stage Xenopus laevis embryos

2016

Self Cite

View full text Add to dashboard Cite

Xenopus laevis is an important model organism in developmental biology. While there is a large literature on changes in the organism’s transcriptome during development, the study of its proteome is at an embryonic state. Several papers have been published recently that characterize the proteome of Xenopus laevis eggs and early stage embryos; however, proteomic sample preparation optimizations have not been reported. Sample preparation is challenging because a large fraction (~90% by weight) of the egg or early stage embryo is yolk. We compared three common protein extraction buffer systems, mammalian Cell-PE LB™ lysing buffer (NP40), sodium dodecyl sulfate (SDS), and 8M urea, in terms of protein extraction efficiency and protein identifications. SDS extracts contained the highest concentration of proteins, but this extract was dominated by a high concentration of yolk proteins. In contrast, NP40 extracts contained ~30% of the protein concentration as SDS extracts, but excelled in discriminating against yolk proteins, which resulted in more protein and peptide identifications. We then compared digestion methods using both SDS and NP40 extraction methods with one-dimensional reverse phase liquid chromatography tandem mass spectrometry (RPLC-MS/MS). NP40 coupled to a filter-aided sample preparation (FASP) procedure produced nearly twice the number of protein and peptide identifications compared to alternatives. When NP40-FASP samples were subjected to two-dimensional RPLC-ESI-MS/MS, a total of 5171 proteins and 38,885 peptides were identified from single stage of embryos (stage 2), increasing the number of protein identifications by 23% in comparison to other traditional protein extraction methods.

show abstract

“…SDS, NP‐40, RapiGest, as well as urea and ammonium bicarbonate used for solubilization of islet secretomes in a bottom‐up approach, was performed by Schmudlach et al. . These secretomes are proteins secreted by a cell or organ in people with diabetes.…”

Section: Sds Removalmentioning

confidence: 99%

Sample Clean‐up Strategies for ESI Mass Spectrometry Applications in Bottom‐up Proteomics: Trends from 2012 to 2016

2017

View full text Add to dashboard Cite

Bottom-up proteomics is a mass spectrometric (MS)-based approach for the characterization of peptides obtained from in-solution protein digestion. MS is favored over other methods for peptide and protein analysis because of its better sensitivity and high throughput. Inorganic ions and surfactants present in the sample or produced during tryptic digestion are detrimental in MS analysis and affect the proteome data, thus sample preparation for removal of these unwanted components has become essential. Here, we review 48 research papers on strategies for removal of salts and surfactants (in particular, SDS) prior to ESI-MS analysis in bottom-up proteomics from 2012 to 2016. The strategies were mostly based on SPE and membrane-based filter-aided sample preparation for salt and SDS removal, respectively. Some known limitations of SPE and filter-aided sample preparation procedures are that they can be time consuming, laborious, and require the use of organic solvents before a concentrated extract suitable for analysis is obtained. The development of faster analytical methods by reducing the sample preparation time and thereby, increasing sample throughput, and in a solvent-less and membrane-less operation, is a significant contribution to proteome research.

show abstract

Sample preparation protocol for bottom-up proteomic analysis of the secretome of the islets of Langerhans

Cited by 18 publications

References 38 publications

Bottom-up proteomics analysis of the secretome of murine islets of Langerhans in elevated glucose levels

Bottom-up proteomics analysis of the secretome of murine islets of Langerhans in elevated glucose levels

Optimization and comparison of bottom-up proteomic sample preparation for early-stage Xenopus laevis embryos

Sample Clean‐up Strategies for ESI Mass Spectrometry Applications in Bottom‐up Proteomics: Trends from 2012 to 2016

Contact Info

Product

Resources

About