Beyond mass spectrometry, the next step in proteomics

Timp, Winston; Timp, G.

doi:10.1126/sciadv.aax8978

Cited by 215 publications

(187 citation statements)

References 166 publications

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“…Although comprehensive and quantitative information on proteins through mass spectrometry can appear more challenging (compared to gene expression or antibody-based assays), high-throughput proteomic techniques are greatly needed for deciphering the protein make-up of MSCs as well as quantifying the changes in physiological or pathological conditions. New technological and bioinformatics advances in mass spectrometry bring this closer to reality [132]. Moreover, the development of standardized procedures for large-scale clinical-grade secretome-based products and their manufacturing, which is highly needed for future clinical trials, remains insufficient.…”

Section: Resultsmentioning

confidence: 99%

Paracrine Mechanisms of Mesenchymal Stromal Cells in Angiogenesis

Maacha

Sidahmed²,

Jacob

et al. 2020

Stem Cells International

165

131

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The role of the mesenchymal stromal cell- (MSC-) derived secretome is becoming increasingly intriguing from a clinical perspective due to its ability to stimulate endogenous tissue repair processes as well as its effective regulation of the immune system, mimicking the therapeutic effects produced by the MSCs. The secretome is a composite product secreted by MSC in vitro (in conditioned medium) and in vivo (in the extracellular milieu), consisting of a protein soluble fraction (mostly growth factors and cytokines) and a vesicular component, extracellular vesicles (EVs), which transfer proteins, lipids, and genetic material. MSC-derived secretome differs based on the tissue from which the MSCs are isolated and under specific conditions (e.g., preconditioning or priming) suggesting that clinical applications should be tailored by choosing the tissue of origin and a priming regimen to specifically correct a given pathology. MSC-derived secretome mediates beneficial angiogenic effects in a variety of tissue injury-related diseases. This supports the current effort to develop cell-free therapeutic products that bring both clinical benefits (reduced immunogenicity, persistence in vivo, and no genotoxicity associated with long-term cell cultures) and manufacturing advantages (reduced costs, availability of large quantities of off-the-shelf products, and lower regulatory burden). In the present review, we aim to give a comprehensive picture of the numerous components of the secretome produced by MSCs derived from the most common tissue sources for clinical use (e.g., AT, BM, and CB). We focus on the factors involved in the complex regulation of angiogenic processes.

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Section: Resultsmentioning

confidence: 99%

Paracrine Mechanisms of Mesenchymal Stromal Cells in Angiogenesis

Maacha

Sidahmed²,

Jacob

et al. 2020

Stem Cells International

165

131

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“…Third, while nanopores play an important role in Stage 2 in the proposed approach that role is not a central one. Such properties as pore length, electro-osmotic effects, hydrophobicity, and others that affect conventional nanopore-based sequencing [4] are not important. The only major consideration is that the pore diameter be sufficiently small that a tRNA (charged or uncharged) or AARS cannot enter the pore.…”

Section: Resultsmentioning

confidence: 99%

“…Often test samples may contain only trace amounts so single molecule methods are of interest. With nanopores a single polymer molecule (DNA, RNA, protein) is electrophoretically transported through a pore in an electrolytic cell (e-cell) and the resulting current blockades probed to identify the sequence of monomers in the polymer [3,4]. A comprehensive review that charts developments in protein sequencing along four different directions is given in [5].…”

Section: Introductionmentioning

confidence: 99%

“…The in vivo error rate without error correction is about 1 in 250, with error correction about 1 in 10000 [12]. As a result the extreme precision required with nanopores to measure blockade current levels due to 20 different monomer types [4], many differing only marginally from others, is no longer required. In the present case it is sufficient to distinguish the blockade level of any amino acid from the significantly larger one due to AMP (adenosine monophosphate), and of AMP from that of ATP.…”

Section: Introductionmentioning

confidence: 99%

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Label-free amino acid identification for de novo protein sequencing via tRNA charging and current blockade in a nanopore

Sampath¹

2020

Preprint

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Label-free identification of amino acids (AAs) based on superspecific transfer RNAs (tRNAs) and amino-acyl tRNA synthetase (AARS) is explored. Molecules of a specific AA, a tRNA and its cognate AARS, and adenosine triphosphate (ATP) from a reservoir are confined to a micro-sized cavity by hydraulic pressure to counter the effects of diffusion. In this confined space a cognate tRNA in the cavity gets charged with AA and adenosine monophosphate (AMP) is released. The products are transferred to an electrolytic cell with a nanopore where AA, AMP, and ATP cause current blockades of different sizes. If an AMP-sized blockade occurs it means that a tRNA molecule has been charged with AA, this identifies AA; otherwise the procedure is repeated with a different tRNA and AARS until AA is identified. Unlike in most other nanopore-based methods, there is no need for precise measurement of current blockade levels. The efficacy of the procedure is assessed by simulating the movement of particles in a reservoir-cavity structure and analyzing the blockades that occur in the nanopore. In the former the probability of reactants escaping the cavity is found to be near 0. In the latter the probability of an error in identifying an AMP blockade is found to be less than 10% in the worst case (which occurs with the largest volume AA, namely Tryptophan). Detailed information and data are provided in a Supplementary File.

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“…It is possible that they were not amenable to or present in sufficient quantity for detection by mass-spectrometry. Western blot with high quality antibodies can be more sensitive than standard bottom-up MS in the detection of certain proteins of low abundance [50]. To overcome this potential limitation, we also employed immunocapture experiments in an attempt to improve sensitivity.…”

Section: Resultsmentioning

confidence: 99%

Protein Interactomes Identify Distinct Pathways forStreptococcus mutansYidC1 and YidC2 Membrane Protein Insertases

Vasquez

Mishra

Kuppuswamy

et al. 2020

Preprint

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20Virulence properties of cariogenic Streptococcus mutans depend on integral membrane proteins. 21Bacterial protein trafficking involves the co-translational signal recognition particle (SRP) 22 pathway components Ffh and FtsY, the SecY translocon, and membrane-localized YidC 23 chaperone/insertases. Unlike Escherichia coli, S. mutans survives loss of the SRP pathway. In 24 addition, S. mutans has two yidC paralogs. The yidC2 phenotype largely parallels that of ffh 25 and ftsY while the yidC1 phenotype is less severe. This study defined YidC1 and YidC2 26 interactomes to identify their respective functions alone and in concert with the SRP, ribosome, 27 and/or Sec translocon. A chemical cross-linking approach was employed, whereby whole cell 28 lysates were treated with formaldehyde followed by Western blotting using anti-Ffh, FtsY, 29 YidC1 or YidC2 antibodies and mass spectrometry (MS) analysis of gel-shifted bands. Cross-30 linked lysates from WT and yidC2 strains were also reacted with anti-YidC2 antibodies 31 coupled to magnetic Dynabeads TM , with co-captured proteins identified by MS. Additionally, C-32 terminal tails of YidC1 and YidC2 were engineered as glutathione-S-transferase fusion proteins 33 and subjected to 2D Difference Gel Electrophoresis and MS analysis after being reacted with 34 non-cross-linked lysates. Results indicate that YidC2 works in concert with the SRP-pathway, 35 while YidC1 works in concert with the SecY translocon independently of the SRP. In addition, 36 YidC1 and/or YidC2 can act alone in the insertion of a limited number of small integral 37 membrane proteins. The YidC2-SRP and YidC1/SecY pathways appear to function as part of an 38 integrated machinery that couples translation and transport with cell division, as well as 39 transcription and DNA replication. 40 41 42 Importance 43

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Beyond mass spectrometry, the next step in proteomics

Cited by 215 publications

References 166 publications

Paracrine Mechanisms of Mesenchymal Stromal Cells in Angiogenesis

Paracrine Mechanisms of Mesenchymal Stromal Cells in Angiogenesis

Label-free amino acid identification for de novo protein sequencing via tRNA charging and current blockade in a nanopore

Protein Interactomes Identify Distinct Pathways forStreptococcus mutansYidC1 and YidC2 Membrane Protein Insertases

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