A novel LC system embeds analytes in pre-formed gradients for rapid, ultra-robust proteomics

Bache, Nicolai; Geyer, Philipp; Bekker-Jensen, Dorte B.; Hoerning, Ole; Falkenby, Lasse Gaarde; Treit, Peter V.; Doll, Sophia; Paron, Igor; Meier, Florian; Olsen, Jesper V.; Vorm, Ole; Mann, Matthias

doi:10.1101/323048

Cited by 24 publications

(23 citation statements)

References 33 publications

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“…The desalted peptides were dissolved in 50 µl of solvent-A (0.1% formic acid in water) prior to loading on to Evotips (https://www.evosep.com/) as described in [62]. Briefly, the Evotips are activated by incubating in 100% isopropanol and then washed twice with 20 µl of Solvent-B (100% Acetonitrile in 0.1% formic acid by volume) and subsequently equilibrated by washing twice with 20 µl of solvent-A (0.1% formic acid in water).…”

Section: Lc-ms/ms Analysismentioning

confidence: 99%

PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins

Berndsen

Lis

Yeshaw

et al. 2019

Preprint

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Mutations that activate LRRK2 protein kinase cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif controlling interaction with effectors. An siRNA screen of all protein phosphatases revealed that a poorly studied protein phosphatase, PPM1H, counteracts LRRK2 signaling by specifically dephosphorylating Rab proteins. PPM1H knock out increased endogenous Rab phosphorylation and inhibited Rab dephosphorylation. Overexpression of PPM1H suppressed LRRK2-mediated Rab phosphorylation. PPM1H also efficiently and directly dephosphorylated Rab8A in biochemical studies. A "substrate-trapping" PPM1H mutant (Asp288Ala) binds with high affinity to endogenous, LRRK2-phosphorylated Rab proteins, thereby blocking dephosphorylation seen upon addition of LRRK2 inhibitors. PPM1H is localized to the Golgi and its knockdown suppresses primary cilia formation, similar to pathogenic LRRK2. Thus, PPM1H acts as a key modulator of LRRK2 signaling by controlling dephosphorylation of Rab proteins. PPM1H activity enhancers could offer a new therapeutic approach to prevent or treat Parkinson's disease.

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Section: Lc-ms/ms Analysismentioning

confidence: 99%

PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins

Berndsen

Lis

Yeshaw

et al. 2019

Preprint

View full text Add to dashboard Cite

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“…Technological advancements in instrumentation, coupling to mass spectrometer and improvements in separation performance have accompanied this development [4,27]. Even though nanoLC-MS is still the most commonly used analysis platform for advanced proteomics [28,29], several studies have demonstrated that combination of nanoLC-MS and CE-MS methods increase the number of identified proteins as they are orthogonal and provide complementary results [30].…”

Section: Introductionmentioning

confidence: 99%

Sodium dodecyl sulfate free gel electrophoresis/electroelution sorting for peptide fractionation

Ramos

González

Sosa‐Acosta

et al. 2019

J of Separation Science

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Shotgun proteomics based on peptide fractionation by using liquid chromatography has become the common procedure for proteomic studies, although in the very beginning of the field, protein separation by using electrophoresis was the main tool. Nonetheless, during the last two decades, the electrophoretic techniques for peptide mixtures fractionation have evolved as a result of relevant technological improvements. We also proposed the combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis for protein fractionation and sodium dodecyl sulfate free polyacrylamide gel electrophoresis for peptide separation as a novel procedure for proteomic studies. Here, we present an optimized device for sodium dodecyl sulfate free polyacrylamide gel electrophoresis improving peptide recoveries respect to the established electrophoretic technique off gel electrophoresis meanwhile conserving the excellent resolution described for the former technique in slab gel based systems. The device simultaneously allows the separation and the collection of fractionated peptides in solution.

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“…However, most of these approaches fail to achieve the meaningful depth of quantitative proteome coverage within short experimental times. Many of the recent efforts were focused on hybrid experimental pipelines, in which MS/MS-based identification is combined with MS1-based quantitation 3,16 .…”

mentioning

confidence: 99%

DirectMS1: MS/MS-free identification of 1000 proteins of cellular proteomes in 5 minutes

Ivanov

Bubis

Gorshkov

et al. 2019

Preprint

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Proteome characterization relies heavily on tandem mass spectrometry (MS/MS) and is thus associated with instrumentation complexity, lengthy analysis time, and limited duty-cycle. Itwas always tempting to implement approaches which do not require MS/MS, yet, they were constantly failing in achieving meaningful depth of quantitative proteome coverage within short experimental times, which is particular important for clinical or biomarker discovery applications. Here, we report on the first successful attempt to develop a truly MS/MS-free and label-free method for bottom-up proteomics. We demonstrate identification of 1000 protein groups for a standard HeLa cell line digest using 5-minute LC gradients. The amount of loaded sample was varied in a range from 1 ng to 500 ng, and the method demonstrated 10-fold higher sensitivity compared with the standard MS/MS-based approach. Due to significantly higher sequence coverage obtained by the developed method, it outperforms all popular MS/MSbased label-free quantitation approaches.Advances in mass-spectrometry-based proteomic technologies resulted in dramatically increased depth, throughput, and sensitivity of proteome coverage. Up to 10,000 proteins can be identified in an 100 minute analysis of human cell proteomes using state-of-the-art high-resolution Orbitrap mass spectrometry 1 . Recently, the notable trend in LC-MS technology developments has been toward increasing the throughput of the proteome-wide analysis, while preserving the quantitation accuracy 2,3 .However, these achievements rely heavily on the use of tandem mass spectrometry (MS/MS), which includes sequential isolation of eluting peptides followed by their fragmentation. While being a crucial and seemingly the only source of sequence-specific information about the peptides, MS/MS brings a number of well-known challenges. Due to the limited both the speed of the mass analyzer (which is

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A novel LC system embeds analytes in pre-formed gradients for rapid, ultra-robust proteomics

Cited by 24 publications

References 33 publications

PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins

PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins

Sodium dodecyl sulfate free gel electrophoresis/electroelution sorting for peptide fractionation

DirectMS1: MS/MS-free identification of 1000 proteins of cellular proteomes in 5 minutes

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