Well-Plate μFASP for Proteomic Analysis of Single Pancreatic Islets

Sandbaumhüter, Friederike Andrea; Nezhyva, Mariya; Eriksson, Olle; Engberg, Adam; Kreuger, Johan; Andrén, Per E.; Jansson, Erik T.

doi:10.1021/acs.jproteome.2c00047

Cited by 8 publications

(14 citation statements)

References 27 publications

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“…Peptic peptides were sequenced with PLGS 3.0.3 (Waters Corporation, Manchester, United Kingdom), as previously described, , with a modification of setting enzymes to “non-specific.” CE–MS data obtained with UDMS E were searched against a database of UNIPROT peptide sequence entries for HBA_BOVIN, HBB_BOVIN, and PEPA_PIG. Complete data were further processed with the HX-DEAL module in Mass Spec Studio for HDX-analysis …”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…The instrument was calibrated on the day of use using an appropriate dilution of sodium iodide (18600791-3, Waters Corporation, Manchester, United Kingdom) in 50% MeOH and 0.1% formic acid delivered to the electrospray emitter at 500 nL/min through the sheath liquid capillary. Unlabeled peptides were analyzed in data independent acquisition (DIA) mode using the UDMS E method with instrument settings previously reported for this instrument, unless otherwise stated. , …”

Section: Methodsmentioning

confidence: 99%

“…Unlabeled peptides were analyzed in data independent acquisition (DIA) mode using the UDMS E method with instrument settings previously reported for this instrument, unless otherwise stated. 36,37 Hydrogen-Deuterium Exchange Sample Preparation and Workflow. For HDX−MS, stock solutions of either 2 mg/mL HPLC peptide standard, 4.4 mM ATII with 4.4 mM BK or 120 mg/mL intact bovine Hb were prepared in 50 mM ammonium bicarbonate in H 2 O, pH 6.79.…”

Section: T H Imentioning

confidence: 99%

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Zero-Degree Celsius Capillary Electrophoresis Electrospray Ionization for Hydrogen Exchange Mass Spectrometry

2022

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Currently, fast liquid chromatographic separations at low temperatures are exclusively used for the separation of peptides generated in hydrogen deuterium exchange (HDX) workflows. However, it has been suggested that capillary electrophoresis may be a better option for use with HDX. We performed in solution HDX on peptides and bovine hemoglobin (Hb) followed by quenching, pepsin digestion, and cold capillary electrophoretic separation coupled with mass spectrometry (MS) detection for benchmarking a laboratory-built HDX–MS platform. We found that capillaries with a neutral coating to eliminate electroosmotic flow and adsorptive processes provided fast separations with upper limit peak capacities surpassing 170. In contrast, uncoated capillaries achieved 30% higher deuterium retention for an angiotensin II peptide standard owing to faster separations but with only half the peak capacity of coated capillaries. Data obtained using two different separation conditions on peptic digests of Hb showed strong agreement of the relative deuterium uptake between methods. Processed data for denatured versus native Hb after deuterium labeling for the longest timepoint in this study (50,000 s) also showed agreement with subunit interaction sites determined by crystallographic methods. All proteomic data are available under DOI: 10.6019/PXD034245.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: T H Imentioning

confidence: 99%

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Zero-Degree Celsius Capillary Electrophoresis Electrospray Ionization for Hydrogen Exchange Mass Spectrometry

2022

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“…The authors have also developed a 96-well plate version of the micro-FASP method, which showed high reproducibility (R 2 > 0.99) when processing Xenopus laevis lysate (12 replicates, 1 μg each). Similarly, Sandbaumhüter et al developed a flexible well-plate μFASP device suitable for a small amount of protein (1 μg) [ 113 ]. The filter area for the centrifugal filter units of the conventional FASP was reduced from approximately 119 mm 2 to 0.785 mm 2 for the μFASP device.…”

Section: From Proteins To Peptidesmentioning

confidence: 99%

Bottom-Up Proteomics: Advancements in Sample Preparation

Duong

Lee

2023

IJMS

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Liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based proteomics is a powerful technique for profiling proteomes of cells, tissues, and body fluids. Typical bottom-up proteomic workflows consist of the following three major steps: sample preparation, LC–MS/MS analysis, and data analysis. LC–MS/MS and data analysis techniques have been intensively developed, whereas sample preparation, a laborious process, remains a difficult task and the main challenge in different applications. Sample preparation is a crucial stage that affects the overall efficiency of a proteomic study; however, it is prone to errors and has low reproducibility and throughput. In-solution digestion and filter-aided sample preparation are the typical and widely used methods. In the past decade, novel methods to improve and facilitate the entire sample preparation process or integrate sample preparation and fractionation have been reported to reduce time, increase throughput, and improve reproducibility. In this review, we have outlined the current methods used for sample preparation in proteomics, including on-membrane digestion, bead-based digestion, immobilized enzymatic digestion, and suspension trapping. Additionally, we have summarized and discussed current devices and methods for integrating different steps of sample preparation and peptide fractionation.

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Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling

Sandbaumhüter,

Nezhyva,

Andrén

et al. 2023

Anal. Chem.

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Thermal proteome profiling with label-free quantitation using ion-mobility-enhanced LC–MS offers versatile data sets, providing information on protein differential expression, thermal stability, and the activities of transcription factors. We developed a multidimensional data analysis workflow for label-free quantitative thermal proteome profiling (TPP) experiments that incorporates the aspects of gene set enrichment analysis, differential protein expression analysis, and inference of transcription factor activities from LC–MS data. We applied it to study the signaling processes downstream of melanocortin 3 receptor (MC3R) activation by endogenous agonists derived from the proopiomelanocortin prohormone: ACTH, α-MSH, and γ-MSH. The obtained information was used to map signaling pathways downstream of MC3R and to deduce transcription factors responsible for cellular response to ligand treatment. Using our workflow, we identified differentially expressed proteins and investigated their thermal stability. We found in total 298 proteins with altered thermal stability, resulting from MC3R activation. Out of these, several proteins were transcription factors, indicating them as being downstream target regulators that take part in the MC3R signaling cascade. We found transcription factors CCAR2, DDX21, HMGB2, SRSF7, and TET2 to have altered thermal stability. These apparent target transcription factors within the MC3R signaling cascade play important roles in immune responses. Additionally, we inferred the activities of the transcription factors identified in our data set. This was done with Bayesian statistics using the differential expression data we obtained with label-free quantitative LC–MS. The inferred transcription factor activities were validated in our bioinformatic pipeline by the phosphorylated peptide abundances that we observed, highlighting the importance of post-translational modifications in transcription factor regulation. Our multidimensional data analysis workflow allows for a comprehensive characterization of the signaling processes downstream of MC3R activation. It provides insights into protein differential expression, thermal stability, and activities of key transcription factors. All proteomic data generated in this study are publicly available at DOI: 10.6019/PXD039945.

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Well-Plate μFASP for Proteomic Analysis of Single Pancreatic Islets

Cited by 8 publications

References 27 publications

Zero-Degree Celsius Capillary Electrophoresis Electrospray Ionization for Hydrogen Exchange Mass Spectrometry

Zero-Degree Celsius Capillary Electrophoresis Electrospray Ionization for Hydrogen Exchange Mass Spectrometry

Bottom-Up Proteomics: Advancements in Sample Preparation

Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling

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