Characterization of a high field Orbitrap mass spectrometer for proteome analysis

Pachl, Fiona; Ruprecht, Benjamin; Lemeer, Simone; Küster, Bernhard

doi:10.1002/pmic.201300076

Cited by 10 publications

(8 citation statements)

References 47 publications

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“…The analysis using the Hybrid LTQ-Orbitrap Velos type of instrument was performed as previously described. 46 The instrument was operated in data-dependent acquisition mode to automatically switch between MS and MS/MS. MS/MS spectra of these ions were obtained by fragmenting the ions using collision-induced dissociation with normalized collision energy of 30% in the ion trap.…”

Section: Methodsmentioning

confidence: 99%

SIRT1 protects the heart from ER stress-induced cell death through eIF2α deacetylation

et al. 2016

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Over the past decade, endoplasmic reticulum (ER) stress has emerged as an important mechanism involved in the pathogenesis of cardiovascular diseases including heart failure. Cardiac therapy based on ER stress modulation is viewed as a promising avenue toward effective therapies for the diseased heart. Here, we tested whether sirtuin-1 (SIRT1), a NAD-dependent deacetylase, participates in modulating ER stress response in the heart. Using cardiomyocytes and adult-inducible SIRT1 knockout mice, we demonstrate that SIRT1 inhibition or deficiency increases ER stress-induced cardiac injury, whereas activation of SIRT1 by the SIRT1-activating compound STAC-3 is protective. Analysis of the expression of markers of the three main branches of the unfolded protein response (i.e., PERK/eIF2α, ATF6 and IRE1) showed that SIRT1 protects cardiomyocytes from ER stress-induced apoptosis by attenuating PERK/eIF2α pathway activation. We also present evidence that SIRT1 physically interacts with and deacetylates eIF2α. Mass spectrometry analysis identified lysines K141 and K143 as the acetylation sites on eIF2α targeted by SIRT1. Furthermore, mutation of K143 to arginine to mimic eIF2α deacetylation confers protection against ER stress-induced apoptosis. Collectively, our findings indicate that eIF2α deacetylation on lysine K143 by SIRT1 is a novel regulatory mechanism for protecting cardiac cells from ER stress and suggest that activation of SIRT1 has potential as a therapeutic approach to protect the heart against ER stress-induced injury.

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Section: Methodsmentioning

confidence: 99%

SIRT1 protects the heart from ER stress-induced cell death through eIF2α deacetylation

et al. 2016

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“…Individually 3476 protein groups were found using Elite CID and 3985 by Q Exactive HCD. Others have found a slight advantage for Orbitrap Elite CID analysis of complex lysates (29,30). The small increase of 170 protein groups when CID data is added to HCD shows there is little reason to use both instruments for tryptic work.…”

Section: Methodsmentioning

confidence: 99%

Confetti: A Multiprotease Map of the HeLa Proteome for Comprehensive Proteomics

Guo

Trudgian

Lemoff

et al. 2014

Molecular & Cellular Proteomics

133

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Bottom-up proteomics largely relies on tryptic peptides for protein identification and quantification. Tryptic digestion often provides limited coverage of protein sequence because of issues such as peptide length, ionization efficiency, and post-translational modification colocalization. Unfortunately, a region of interest in a protein, for example, because of proximity to an active site or the presence of important post-translational modifications, may not be covered by tryptic peptides. Detection limits, quantification accuracy, and isoform differentiation can also be improved with greater sequence coverage. Selected reaction monitoring (SRM) would also greatly benefit from being able to identify additional targetable sequences. In an attempt to improve protein sequence coverage and to target regions of proteins that do not generate useful tryptic peptides, we deployed a multiprotease strategy on the HeLa proteome. First, we used seven commercially available enzymes in single, double, and triple enzyme combinations. A total of 48 digests were performed. 5223 proteins were detected by analyzing the unfractionated cell lysate digest directly; with 42% mean sequence coverage. Additional strong-anion exchange fractionation of the most complementary digests permitted identification of over 3000 more proteins, with improved mean sequence coverage. We then constructed a web application (https://proteomics.swmed.edu/confetti) that allows the community to examine a target protein or protein isoform in order to discover the enzyme or combination of enzymes that would yield peptides spanning a certain region of interest in the sequence. Finally, we examined the use of nontryptic digests for SRM. From our strong-anion exchange fractionation data, we were able to identify three or more proteotypic SRM candidates within a single digest for 6056 genes. Surprisingly, in 25% of these cases the digest producing the most observable proteotypic peptides was neither trypsin nor Lys-C.

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“…A total of 100 μg protein from each pool was used for three technical replicates. Proteins were digested by the FASP approach (Wisniewski et al., ) and the resulting peptides were fractionated by strong anion exchange stage tips prior to nano LC‐MS/MS analysis on an Eksigent nanoLC‐Ultra 1D+ (Eksigent, Dublin, CA) coupled to an Orbitrap Elite mass spectrometer (Thermo Scientific, Bremen, Germany) (Pachl et al., ). Peptides were delivered to a trap column (100 μm × 2 cm, packed in‐house with Reprosil‐Pur C18‐AQ 5 μm resin, Dr. Maisch, Ammerbuch, Germany) at a flow rate of 5 μL/min in 100% solvent A (0.1% formic acid in HPLC grade water).…”

Section: Methodsmentioning

confidence: 99%

Reduced mitochondrial mass and function add to age-related susceptibility toward diet-induced fatty liver in C57BL/6J mice

et al. 2016

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Nonalcoholic fatty liver disease (NAFLD) is a major health burden in the aging society with an urging medical need for a better understanding of the underlying mechanisms. Mitochondrial fatty acid oxidation and mitochondrial‐derived reactive oxygen species (ROS) are considered critical in the development of hepatic steatosis, the hallmark of NAFLD. Our study addressed in C57BL/6J mice the effect of high fat diet feeding and age on liver mitochondria at an early stage of NAFLD development. We therefore analyzed functional characteristics of hepatic mitochondria and associated alterations in the mitochondrial proteome in response to high fat feeding in adolescent, young adult, and middle‐aged mice. Susceptibility to diet‐induced obesity increased with age. Young adult and middle‐aged mice developed fatty liver, but not adolescent mice. Fat accumulation was negatively correlated with an age‐related reduction in mitochondrial mass and aggravated by a reduced capacity of fatty acid oxidation in high fat‐fed mice. Irrespective of age, high fat diet increased ROS production in hepatic mitochondria associated with a balanced nuclear factor erythroid‐derived 2 like 2 (NFE2L2) dependent antioxidative response, most likely triggered by reduced tethering of NFE2L2 to mitochondrial phosphoglycerate mutase 5. Age indirectly influenced mitochondrial function by reducing mitochondrial mass, thus exacerbating diet‐induced fat accumulation. Therefore, consideration of age in metabolic studies must be emphasized.

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Characterization of a high field Orbitrap mass spectrometer for proteome analysis

Cited by 10 publications

References 47 publications

SIRT1 protects the heart from ER stress-induced cell death through eIF2α deacetylation

SIRT1 protects the heart from ER stress-induced cell death through eIF2α deacetylation

Confetti: A Multiprotease Map of the HeLa Proteome for Comprehensive Proteomics

Reduced mitochondrial mass and function add to age-related susceptibility toward diet-induced fatty liver in C57BL/6J mice

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