A Robust Method for Packing High Resolution C18 RP-nano-HPLC Columns

Jami‐Alahmadi, Yasaman; Pandey, Vijaya; Mayank, Adarsh K.

doi:10.3791/62380

Cited by 30 publications

(25 citation statements)

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“…For liquid chromatography-coupled tandem mass spectrometric (LC-MS/MS) analysis, desalted peptides were fractionated online by nano-flow reversed phase liquid chromatography using a Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific). The column consisted of a 25-cm fused silica capillary with 75 μm inner diameter that was packed in-house 78 with ReproSil-Pur C18 particles (particle size 1.9 μm; pore size 120 Å; Dr. Maisch). The linear 140-min water-acetonitrile gradient from 5% to 35% acetonitrile was delivered at a flow rate of 300 nl/min.…”

Section: Methodsmentioning

confidence: 99%

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Genome-wide base editor screen identifies regulators of protein abundance in yeast

Schubert

Bloom

Sadhu

et al. 2022

Preprint

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Abundance of proteins is extensively regulated both transcriptionally and post-transcriptionally. To systematically characterize how regulation of protein abundance is encoded in the genome and identify protein regulators on a genome-wide scale, we developed a genetic screen that uses a CRISPR base editor. We examined the effects of 16,452 genetic perturbations on the abundance of eleven yeast proteins representing a variety of cellular functions. Thereby, we uncovered hundreds of regulatory relationships, including a novel link between the GAPDH isoenzymes Tdh1/2/3 and the Ras/PKA pathway. Many of the identified regulators are specific to one of the eleven proteins, but we also found genes that, upon perturbation, affected the abundance of most of the tested proteins. While the more specific regulators usually act transcriptionally, broad regulators often have roles in protein translation. Our results provide unprecedented insights into the components, scale and connectedness of the protein regulatory network.

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Section: Methodsmentioning

confidence: 99%

“…The column consisted of a 25-cm fused silica capillary with 75 μm inner diameter that was packed in-house 78 with ReproSil-Pur C18 particles (particle size 1.9 μm; pore size 120 Å; Dr. Maisch). The linear 140-min water-acetonitrile gradient from 5% to 35% acetonitrile was delivered at a flow rate of 300 nl/min.…”

Section: Lc-ms Proteomics Data Acquisitionmentioning

confidence: 99%

Genome-wide base editor screen identifies regulators of protein abundance in yeast

Schubert

Bloom

Sadhu

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…After quenching the digest by the addition of formic acid to 5% (v/v), peptides were desalted using Pierce C18 tips (Thermo Fisher Scientific), dried by vacuum centrifugation, and resuspended in 5% formic acid. Peptides were fractionated online using reversed phase chromatography on in-house packed C18 columns as described before (64). The 140 min gradient of increasing acetonitrile was delivered using a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific).…”

Section: Protein Identification By Mass Spectrometrymentioning

confidence: 99%

The Nse5/6-like SIMC1-SLF2 Complex Localizes SMC5/6 to Viral Replication Centers

Oravcová

Nie

Zilio

et al. 2022

Preprint

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The human SMC5/6 complex is a conserved guardian of genome stability and an emerging component of antiviral responses. These disparate functions likely require distinct mechanisms of SMC5/6 regulation. In yeast, Smc5/6 is regulated by its Nse5/6 subunits, but such regulatory subunits for human SMC5/6 are poorly defined. Here, we identify a novel SMC5/6 subunit called SIMC1 that contains SUMO interacting motifs (SIMs) and an Nse5-like domain. We isolated SIMC1 from the proteomic environment of SMC5/6 within polyomavirus large T antigen (LT)-induced subnuclear compartments. SIMC1 uses its SIMs and Nse5- like domain to localize SMC5/6 to polyomavirus replication centers (PyVRCs) at SUMO-rich PML nuclear bodies. SIMC1's Nse5-like domain binds to the putative Nse6 orthologue SLF2 to form an anti-parallel helical dimer resembling the yeast Nse5/6 structure. SIMC1-SLF2 structure-based mutagenesis defines a conserved surface region containing the N-terminus of SIMC1's helical domain that regulates SMC5/6 localization to PyVRCs. Furthermore, SLF1, which recruits SMC5/6 to DNA lesions, binds SLF2 analogously to SIMC1 and forms a distinct Nse5/6-like complex. Thus, two Nse5/6-like complexes independently regulate human SMC5/6: SIMC1-SLF2 responding to viral challenge and SLF1/2 recognizing DNA damage.

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“…77 Digested peptides were resuspended in 5% formic acid and fractionated online using a 25cm long, 75 μM inner diameter fused silica capillary packed in-house with bulk C18 reversed phase resin (length, 25 cm; inner diameter, 75 μM; particle size, 1.9 μm; pore size, 100 Å; Dr. Maisch GmbH). 78 The 140-minute water-acetonitrile gradient was delivered using a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific) at a flow rate of 300 nL/min (Buffer A: water with 3% DMSO and 0.1% formic acid and Buffer B: acetonitrile with 3% DMSO and 0.1% formic acid). Fractionated peptides were ionized and analyzed by tandem mass spectrometry (MS/MS) Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific).…”

Section: Mass Spectrometry (Ms)mentioning

confidence: 99%

Substrate trapping approach identifies TRIM25 ubiquitination targets involved in diverse cellular and antiviral processes

Yang

Huang

Jami‐Alahmadi

et al. 2022

Preprint

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The tripartite motif (TRIM) family of E3 ubiquitin ligases is well known for its roles in antiviral restriction and innate immunity regulation, in addition to many other cellular pathways. In particular, TRIM25-mediated ubiquitination affects both carcinogenesis and antiviral response. While individual substrates have been identified for TRIM25, it remains unclear how it regulates diverse processes. Here we characterized a mutation, R54P, critical for TRIM25 catalytic activity, which we successfully utilized to "trap" substrates. We demonstrated that TRIM25 targets proteins implicated in stress granule formation (G3BP1/2), nonsense-mediated mRNA decay (UPF1), and nucleoside synthesis (NME1). R54P abolishes TRIM25 inhibition of alphaviruses independently of the host interferon response, suggesting that this antiviral effect is a direct consequence of ubiquitination. Consistent with that, we observed diminished antiviral activity upon knockdown of several TRIM25-R54P specific interactors including NME1. Our findings highlight that multiple substrates mediate the cellular and antiviral activities of TRIM25, illustrating the multi-faceted role of this ubiquitination network in diverse biological processes.

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A Robust Method for Packing High Resolution C18 RP-nano-HPLC Columns

Cited by 30 publications

References 0 publications

Genome-wide base editor screen identifies regulators of protein abundance in yeast

Genome-wide base editor screen identifies regulators of protein abundance in yeast

The Nse5/6-like SIMC1-SLF2 Complex Localizes SMC5/6 to Viral Replication Centers

Substrate trapping approach identifies TRIM25 ubiquitination targets involved in diverse cellular and antiviral processes

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