Multiplexed Single Ion Mass Spectrometry Improves Measurement of Proteoforms and Their Complexes

Kafader, Jared O.; Melani, Rafael D.; Durbin, Kenneth R.; Ikwuagwu, Bon; Early, Bryan P.; Fellers, Ryan T.; Beu, Steven C.; Zabrouskov, Vlad; Makarov, Alexander; Maze, Joshua T.; Shinholt, Deven L.; Yip, Ping; Tullman‐Ercek, Danielle; Senko, Michael W.; Compton, Philip D.; Kelleher, Neil L.

doi:10.1101/715425

Cited by 7 publications

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“…8a). However, using the new approach of Individual Ion MS (I 2 MS) 21 we were able to precisely profile the mass distribution of these mononucleosomes, centered at ~200 kDa (Supplemental Fig. 8b-d).…”

Section: Figure 1 Three Different Strategies Of Histone Analysis Including the New Approach For Direct Interrogation Of Intact Nucleosomementioning

confidence: 99%

“…http://www.unimod.org/modifications_list.php) as a reference for candidate modifications.MS Data availabilityMS1 , MS2 , and MS 3 spectra presented in the manuscript will be made available online in the MassIVE database under accession code MSV000085238 after acceptance of this work in a peer-reviewed journal.Ion Collection and Data Acquisition for Individual Ion Mass Spectrometry (I 2 MS)Detailed methods for this new technique are reported inKafader, et al (2019) 21. Briefly, this new method uses direct assignment of charge states on individual ions inside an Orbitrap-style mass spectrometer with a harmonic potential.…”

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Decoding the protein composition of whole nucleosomes with Nuc-MS

et al. 2021

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Nuc-MS characterizes histone modifications and variants directly from intact endogenous nucleosomes. Preserving whole nucleosome particles enables precise interrogation of their protein content, as for H3.3-containing nucleosomes which had 6fold co-enrichment of variant H2A.Z over bulk chromatin. Nuc-MS, validated by ChIPseq, showed co-occurrence of oncogenic H3.3K27M with euchromatic marks (e.g., H4K16ac and >15-fold enrichment of H3K79me2). By capturing the entire epigenetic landscape, Nuc-MS provides a new, quantitative readout of nucleosome-level biology. MainThe post-translational modifications (PTMs) decorating the four core histones in an intact nucleosome encode information for nuclear effectors that trigger defined cellular events critical to health and disease. [1][2][3][4] For decades, affinity reagents, mass spectrometry and proteomics have played key roles in characterizing the many histone isoforms and PTMs involved in epigenetic regulation. [5][6][7] However, the standard practice of relying on digestion 8 and/or denaturation 5 removes the linkage between modifications and their nucleosomes of origin (Fig. 1a, at left). By digesting histone mixtures into small peptides, correlations among PTMs are forfeited, precluding strong assertions about the original composition of the intact histone. Similarly, when a nucleosome population is denatured, information about co-localization of histone isoforms and PTMs within the same nucleosome particle is lost. This inference problem diminishes our understanding of the organization and impact of co-occurring modifications, isoforms and mutations in epigenetics and disease pathogenesis. 9,10

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Section: Figure 1 Three Different Strategies Of Histone Analysis Including the New Approach For Direct Interrogation Of Intact Nucleosomementioning

confidence: 99%

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confidence: 99%

Decoding the protein composition of whole nucleosomes with Nuc-MS

et al. 2021

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“…Additional relevant data acquisition parameters were adjusted as follows: mass range: 400-2500 m/z; AGC mode was disabled and the maximum injection time was held constant at 300 ms; enhanced Fourier transform: off; averaging: 0; microscans: 1. Time-domain data files were acquired at detected ion frequencies and recorded as Selective Temporal Overview of Resonant Ions (STORI) files (50). STORI enabled: Enabled.…”

Section: Pims Conditions and Data Acquisitionmentioning

confidence: 99%

“…Charge state (z) is obtained from the slope of induced image current determined by the STORI analysis (50). Accurate charge assignment of each ion was statistically evaluated by comparing the slopes of its isotopologues across different charge states from the entire tissue section.…”

Section: Pims Data Analysis and Image Generationmentioning

confidence: 99%

Direct Imaging and Identification of Proteoforms up to 70 kDa from Human Tissue

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Durbin

et al. 2021

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Imaging of proteoforms in human tissues is hindered by low molecular specificity and limited proteome coverage. Here, we introduce proteoform imaging mass spectrometry (PiMS), which increases the size limit for proteoform detection and identification by 4-fold compared to reported methods, and reveals tissue localization of proteoforms at <80 μm spatial resolution. PiMS advances proteoform imaging by combining liquid sampling (nanospray desorption electrospray ionization, nano-DESI) with ion detection using individual ion mass spectrometry (I2MS). We demonstrate the first proteoform imaging of human kidney, identifying 169 of 400 proteoforms <70 kDa using top-down mass spectrometry and database lookup from the human proteoform atlas, including dozens of key enzymes in primary metabolism. Moreover, PiMS images visualize kidney anatomical structures and cellular neighborhoods in the vasculature versus the medulla or the cortex. The benefits of PiMS are poised to increase proteome coverage for label-free protein imaging of intact tissues.

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“…Recent advances in individual ion mass spectrometry (I 2 MS) have also managed to increase the resolving power (by a factor 20) and an increase of the dynamic range of MS. [ 77 ] Specifically, This I 2 MS allows mass determination in a mixture of denatured proteins or in native complexes with masses ranging from 5 kDa to 3.5 MDa. [ 78 ] However, MS based approaches suffer from significant cost elevations for increasing sensitivity.…”

Section: Other Attempts At Pushing the Limits Of Protein Sequencing Amentioning

confidence: 99%

The Promise of Nanopore Technology: Advances in the Discrimination of Protein Sequences and Chemical Modifications

2020

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ever, only 21 000 distinct protein-coding genes were identified. [2] Meaning just a tiny percentage of DNA has the information necessary for translation of all the proteins present in human cells. Among these genes, only 150 are systematic targets of somatic cancer mutations 2850 are drivers of rare diseases when mutated and ≈1100 are involved in diseases caused by multiple genes. [3] We understand now that mutations are not the only factor responsible for human disease. Epigenetics, another factor of interest responsible for disease, is influenced by several factors such as environment, aging, and lifestyle. One of the major sources of epigenetic changes is protein chemical modification. [4,5] Post-translational modifications consist of proteolytic cleavage or covalent addition of a chemical group to one or more amino acids, altering protein properties. [6] Eukaryote chemical modifications regulate many protein biological functions [7,8] such as apoptosis (cell death), epidermal growth factor receptor signaling, endocytosis, DNAdamage responses, and immunity. These modifications are also involved in a lot of human diseases: [9-11] cancer apparition and dissemination, autoimmune pathologies, neurodegenerative diseases or even type 2 diabetes. These chemical modifications are essential for therapeutic protein manufacturing. [12] More generally, the proteome has a key regulatory role in physiological processes that dictate disease development. [5] An immense number of distinct proteins, at least 10 6 different molecules, resulting from open reading frame (ORFs) variants, splice variants, and functional post-translational modifications generate the basis of the proteome's complexity. Moreover, the fluctuations in protein complexes throughout cell and life cycles and the subcellular localization of distinct proteins add further, often overlooked, complexity to the proteome. [5] We briefly describe the main factors explaining the proteome complexity in Figure 1. Because two-thirds of human genes are estimated to contain one or more alternative spliced exons and a large number of noncoding DNA sequences (introns), the alternative splicing mechanism leads to around 10 5 protein isoforms in eukaryotic cells. [13] This mechanism removes introns and associates combinations of exons together with multiple potential mature transcripts. Another factor increasing the complexity of a cell's protein pool is the variety of chemical modifications they are subject to and their rate. The main Only a small percent of human genomic DNA encodes for proteins. Additionally, protein isoforms variants and chemical modifications are not coded in the genome read by the cell machinery. The resulting protein diversity is deeply involved in regular and diseased cellular processes. One challenge for the field of biotechnology, after human genome sequencing, will be to decipher the proteome at a single molecule scale to analyze single-cell protein variability. In fact, cellular proteic information, often used as a source of biomarkers, is of grea...

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Multiplexed Single Ion Mass Spectrometry Improves Measurement of Proteoforms and Their Complexes

Cited by 7 publications

References 30 publications

Decoding the protein composition of whole nucleosomes with Nuc-MS

Decoding the protein composition of whole nucleosomes with Nuc-MS

Direct Imaging and Identification of Proteoforms up to 70 kDa from Human Tissue

The Promise of Nanopore Technology: Advances in the Discrimination of Protein Sequences and Chemical Modifications

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