A sample preparation workflow for adipose tissue shotgun proteomics and proteogenomics

Khudyakov, Jane; Deyarmin, Jared; Hekman, Ryan; Busqueta, Laura Pujade; Maan, Rasool; Mody, Melony; Banerjee, Reeti; Crocker, Daniel E.; Champagne, Cory D.

doi:10.1242/bio.036731

Cited by 11 publications

(5 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Briefly MS1 spectra were collected by using the Orbitrap mass analyzer; precursor ions were selected via DDA using a quadrupole mass filter and then were fragmented using higher energy collisional dissociation in the collision cell. Instrument and data acquisition settings were the same as reported previously (56), with the exception of the scan range (200 to 1,400 Da), MS1 Orbitrap resolution (120,000), scan time (10 to 130 min), MS/MS Orbitrap resolution (30,000), and MS/MS maximum injection time (150 ms).…”

Section: Methodsmentioning

confidence: 99%

“…To identify any associated contaminants that might have arisen during sample preparation, spectra were searched in the common Repository of Adventitious Proteins (cRAP) (https://www.thegpm.org/crap/index.html). Search parameters were the same as reported previously (56). False-discovery rates for peptide spectral matches and peptides were estimated by searching reversed decoy databases generated from the UniProt/Swiss-Prot and cRAP databases.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Role of BGS13 in the Secretory Mechanism of Pichia pastoris

Naranjo

Jivan

et al. 2019

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

The methylotrophic yeast Pichia pastoris has been utilized for heterologous protein expression for over 30 years. Because P. pastoris secretes few of its own proteins, the exported recombinant protein is the major polypeptide in the extracellular medium, making purification relatively easy. Unfortunately, some recombinant proteins intended for secretion are retained within the cell. A mutant strain isolated in our laboratory, containing a disruption of the BGS13 gene, displayed elevated levels of secretion for a variety of reporter proteins. The Bgs13 peptide (Bgs13p) is similar to the Saccharomyces cerevisiae protein kinase C 1 protein (Pkc1p), but its specific mode of action is currently unclear. To illuminate differences in the secretion mechanism between the wild-type (wt) strain and the bgs13 strain, we determined that the disrupted bgs13 gene expressed a truncated protein that had reduced protein kinase C activity and a different location in the cell, compared to the wt protein. Because the Pkc1p of baker’s yeast plays a significant role in cell wall integrity, we investigated the sensitivity of the mutant strain’s cell wall to growth antagonists and extraction by dithiothreitol, determining that the bgs13 strain cell wall suffered from inherent structural problems although its porosity was normal. A proteomic investigation of the bgs13 strain secretome and cell wall-extracted peptides demonstrated that, compared to its wt parent, the bgs13 strain also displayed increased release of an array of normally secreted, endogenous proteins, as well as endoplasmic reticulum-resident chaperone proteins, suggesting that Bgs13p helps regulate the unfolded protein response and protein sorting on a global scale. IMPORTANCE The yeast Pichia pastoris is used as a host system for the expression of recombinant proteins. Many of these products, including antibodies, vaccine antigens, and therapeutic proteins such as insulin, are currently on the market or in late stages of development. However, one major weakness is that sometimes these proteins are not secreted from the yeast cell efficiently, which impedes and raises the cost of purification of these vital proteins. Our laboratory has isolated a mutant strain of Pichia pastoris that shows enhanced secretion of many proteins. The mutant produces a modified version of Bgs13p. Our goal is to understand how the change in the Bgs13p function leads to improved secretion. Once the Bgs13p mechanism is illuminated, we should be able to apply this understanding to engineer new P. pastoris strains that efficiently produce and secrete life-saving recombinant proteins, providing medical and economic benefits.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Role of BGS13 in the Secretory Mechanism of Pichia pastoris

Naranjo

Jivan

et al. 2019

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

show abstract

“…Recently, using liquid chromatography coupled with advanced tandem mass spectrometry (LC-MS/MS) 31–33 , numerous studies aiming to identify key effectors in the metabolic regulation of adipocytes have reported the results of quantitative proteomic analyses utilising diverse labelling techniques (e.g., isobaric tags for relative and absolute quantification [iTRAQ] 34–39 , tandem mass tags [TMT] 40,41 and stable isotope labelling by amino acids in cell culture [SILAC] 42,43 ). Here, we performed a global quantitative proteomic analysis of six 3T3-L1 cell types (preadipocytes, adipocytes, and co-cultured adipocytes with macrophages in 2D- and 3D-cell culture conditions) using iTRAQ-based 2D-nanoLC-ESI-MS/MS.…”

Section: Introductionmentioning

confidence: 99%

iTRAQ-Based Quantitative Proteomic Comparison of 2D and 3D Adipocyte Cell Models Co-cultured with Macrophages Using Online 2D-nanoLC-ESI-MS/MS

Lee

Park

Kim

et al. 2019

Sci Rep

View full text Add to dashboard Cite

The demand for novel three-dimensional (3D) cell culture models of adipose tissue has been increasing, and proteomic investigations are important for determining the underlying causes of obesity, type II diabetes, and metabolic disorders. In this study, we performed global quantitative proteomic profiling of three 3D-cultured 3T3-L1 cells (preadipocytes, adipocytes and co-cultured adipocytes with macrophages) and their 2D-cultured counterparts using 2D-nanoLC-ESI-MS/MS with iTRAQ labelling. A total of 2,885 shared proteins from six types of adipose cells were identified and quantified in four replicates. Among them, 48 proteins involved in carbohydrate metabolism (e.g., PDHα, MDH1/2, FH) and the mitochondrial fatty acid beta oxidation pathway (e.g., VLCAD, ACADM, ECHDC1, ALDH6A1) were relatively up-regulated in the 3D co-culture model compared to those in 2D and 3D mono-cultured cells. Conversely, 12 proteins implicated in cellular component organisation (e.g., ANXA1, ANXA2) and the cell cycle (e.g., MCM family proteins) were down-regulated. These quantitative assessments showed that the 3D co-culture system of adipocytes and macrophages led to the development of insulin resistance, thereby providing a promising in vitro obesity model that is more equivalent to the in vivo conditions with respect to the mechanisms underpinning metabolic syndromes and the effect of new medical treatments for metabolic disorders.

show abstract

“…Workflow selection and the need for optimization differ depending on whether the downstream analysis focuses on specific protein classes or sample types, or broad coverage of a range of sample types and protein targets. Focused analyses require experiment-specific protocol optimization, and numerous studies describe recommended strategies for isolating and analyzing specific protein subsets. − Here, we conduct a holistic analysis of the organism in order to identify a robust workflow capable of efficiently processing multiple tissue types. To accomplish this, we investigated the effect of homogenization strategy and protein precipitation on protein quantitation using MRM-MS in nine disparate mouse tissues.…”

Section: Introductionmentioning

confidence: 99%

Process and Workflow for Preparation of Disparate Mouse Tissues for Proteomic Analysis

et al. 2020

View full text Add to dashboard Cite

We investigated the effect of homogenization strategy and protein precipitation on downstream protein quantitation using multiple reaction monitoring mass spectrometry (MRM-MS). Our objective was to develop a workflow capable of processing disparate tissue types with high throughput, minimal variability, and maximum purity. Similar abundances of endogenous proteins were measured in nine different mouse tissues regardless of the homogenization method used; however, protein precipitation had strong positive effects on several targets. The best throughput was achieved by lyophilizing tissues to dryness, followed by homogenization via bead-beating without sample buffer. Finally, the effect of tissue perfusion prior to dissection and collection was explored in 20 mouse tissues. MRM-MS showed decreased abundances of blood-related proteins in perfused tissues; however, complete removal was not achieved. Concentrations of nonblood proteins were largely unchanged, although significantly higher variances were observed for proteins from the perfused lung, indicating that perfusion may not be suitable for this organ. We present a simple yet effective tissue processing workflow consisting of harvest of fresh nonperfused tissue, novel lyophilization and homogenization by bead-beating, and protein precipitation. This workflow can be applied to a range of mouse tissues with the advantages of simplicity, minimal manual manipulation of samples, use of commonly available equipment, and high sample quality.

show abstract

A sample preparation workflow for adipose tissue shotgun proteomics and proteogenomics

Cited by 11 publications

References 32 publications

Role of BGS13 in the Secretory Mechanism of Pichia pastoris

Role of BGS13 in the Secretory Mechanism of Pichia pastoris

iTRAQ-Based Quantitative Proteomic Comparison of 2D and 3D Adipocyte Cell Models Co-cultured with Macrophages Using Online 2D-nanoLC-ESI-MS/MS

Process and Workflow for Preparation of Disparate Mouse Tissues for Proteomic Analysis

Contact Info

Product

Resources

About