Miniprep assisted proteomics (MAP) for rapid proteomics sample preparation

Mousseau, C. Bruce; Pierre, Camille; Hu, Daniel D.; Champion, Matthew M.

doi:10.1039/d2ay01549h

Cited by 6 publications

(9 citation statements)

References 55 publications

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“…From Figure 1(b), the in‐gel digestion workflow resulted in the lowest total peptide recovery, though yielded a comparatively higher number of proteins and peptides. Beyond the improved extraction of hydrophobic proteins with SDS, another contributing factor is the removal of interferences prior to digestion [28], which can aid peptide detection. SDS also denatures the proteins, enhancing their digestion and ultimately, peptide detection.…”

Section: Resultsmentioning

confidence: 99%

Assessing the precision of a detergent‐assisted cartridge precipitation workflow for non‐targeted quantitative proteomics

Nickerson,

Gagnon,

Wentzell

et al. 2024

Proteomics

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Detergent‐based workflows incorporating sodium dodecyl sulfate (SDS) necessitate additional steps for detergent removal ahead of mass spectrometry (MS). These steps may lead to variable protein recovery, inconsistent enzyme digestion efficiency, and unreliable MS signals. To validate a detergent‐based workflow for quantitative proteomics, we herein evaluate the precision of a bottom‐up sample preparation strategy incorporating cartridge‐based protein precipitation with organic solvent to deplete SDS. The variance of data‐independent acquisition (SWATH‐MS) data was isolated from sample preparation error by modelling the variance as a function of peptide signal intensity. Our SDS‐assisted cartridge workflow yield a coefficient of variance (CV) of 13%–14%. By comparison, conventional (detergent‐free) in‐solution digestion increased the CV to 50%; in‐gel digestion provided lower CVs between 14% and 20%. By filtering peptides predicting to display lower precision, we further enhance the validity of data in global comparative proteomics. These results demonstrate the detergent‐based precipitation workflow is a reliable approach for in depth, label‐free quantitative proteome analysis.

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Section: Resultsmentioning

confidence: 99%

Assessing the precision of a detergent‐assisted cartridge precipitation workflow for non‐targeted quantitative proteomics

Nickerson,

Gagnon,

Wentzell

et al. 2024

Proteomics

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“…Unless otherwise stated, reagents were obtained from Sigma‐Aldrich (St. Louis, MO). Enriched and cross‐linked protein samples were reduced, alkylated, digested with trypsin (Promega, Madison, WI) essentially as described earlier (Anderson et al, 2022; Avila‐Cobian et al, 2022; Mousseau et al, 2023), and analyzed in technical triplicates on a QE‐HF mass spectrometer (Thermo Fisher Scientific, Walthman, MA) coupled to a nano‐LC (Waters, Billerica, MA) running a TOP15 Data‐dependent method. Database searching and label‐free quantification (LFQ) of RAW files was performed against the PAO1 FASTA genome database concatenated with common contaminants, as obtained from the Pseudomonas Genome DB (the Cystic Fibrosis Foundation, Therapeutics) using MaxQuant as in Avila‐Cobian et al (Avila‐Cobian et al, 2022).…”

Section: Methodsmentioning

confidence: 99%

“…ChemiDoc Imaging System (Bio‐Rad, Hercules, CA) was used for obtaining the images of fluorescence and western blots. N ζ ‐[(2‐azidoethoxy)carbonyl]‐ l ‐lysine (compound 1 ) was synthesized according to previous reports (De Ruysscher et al, 2020; Mousseau et al, 2023). Scheme S2 shows the synthesis of compounds 1 , 3 , and 4 .…”

Section: Methodsmentioning

confidence: 99%

Amber‐codon suppression for spatial localization and in vivo photoaffinity capture of the interactome of the Pseudomonas aeruginosa rare lipoprotein A lytic transglycosylase

Avila‐Cobian,

Hoshino,

Horsman

et al. 2023

Protein Science

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The 11 lytic transglycosylases of Pseudomonas aeruginosa have overlapping activities in the turnover of the cell‐wall peptidoglycan. Rare lipoprotein A (RlpA) is distinct among the 11 by its use of only peptidoglycan lacking peptide stems. The spatial localization of RlpA and its interactome within P. aeruginosa are unknown. We employed suppression of introduced amber codons at sites in the rlpA gene for the introduction of the unnatural‐amino‐acids Νζ‐[(2‐azidoethoxy)carbonyl]‐l‐lysine (compound 1) and Nζ‐[[[3‐(3‐methyl‐3H‐diazirin‐3‐yl)propyl]amino]carbonyl]‐l‐lysine (compound 2). In live P. aeruginosa, full‐length RlpA incorporating compound 1 into its sequence was fluorescently tagged using strained‐promoted alkyne‐azide cycloaddition and examined by fluorescence microscopy. RlpA is present at low levels along the sidewall length of the bacterium, and at higher levels at the nascent septa of replicating bacteria. In intact P. aeruginosa, UV photolysis of full‐length RlpA having compound 2 within its sequence generated a transient reactive carbene, which engaged in photoaffinity capture of neighboring proteins. Thirteen proteins were identified. Three of these proteins—PBP1a, PBP5, and MreB—are members of the bacterial divisome. The use of the complementary methodologies of non‐canonical amino‐acid incorporation, photoaffinity proximity analysis, and fluorescent microscopy confirm a dominant septal location for the RlpA enzyme of P. aeruginosa, as a divisome‐associated activity. This accomplishment adds to the emerging recognition of the value of these methodologies for identification of the intracellular localization of bacterial proteins.

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“…The mass-spectrometry methodology and analysis on the photoaffinity-captured Slt samples are similarly described in Avila-Cobian et al (2023). Briefly, enriched and crosslinked Slt protein samples were precipitated with acetone, reduced, alkylated, and digested with trypsin (Promega, Madison, WI) on S-Trap Micros (Protifi, Fairport, NY) by previously described methodologies (Andersen et al, 2022;Avila-Cobian et al, 2022;Mousseau et al, 2023). The samples were analyzed in technical triplicates on a tim-sTOF Pro (Bruker Scientific, Billerica, MA) coupled to an Evosep One (Evosep, Odense, Denmark) running a Whis-per20 LC method.…”

Section: Mass-spectrometry and Proteomics Analysismentioning

confidence: 99%

Lytic transglycosylase Slt of Pseudomonas aeruginosa as a periplasmic hub protein

Avila‐Cobian,

De Benedetti,

Hoshino

et al. 2024

Protein Science

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Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber‐codon‐suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber‐codon‐suppression methodology was also used to incorporate a photoaffinity amino acid for the capture of partner proteins. Mass‐spectrometry‐based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the protein partners of Slt span disparate periplasmic protein families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (KD < 0.5 μM) include PBPs (PBP1a, KD = 0.07 μM; PBP5 = 0.4 μM); other lytic transglycosylases (SltB2, KD = 0.09 μM; RlpA, KD = 0.4 μM); a type VI secretion system effector (Tse5, KD = 0.3 μM); and a regulatory protease for alginate biosynthesis (AlgO, KD < 0.4 μM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub protein within the periplasm.

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Miniprep assisted proteomics (MAP) for rapid proteomics sample preparation

Abstract: Miniprep Assisted Proteomics (MAP) is a rapid approach to bottom-up proteomics sample preparation by adventitious binding to Si-DNA minipreps. This combines the consistency of a commercially produced column with the low-cost of in-house devices.

Cited by 6 publications

References 55 publications

Assessing the precision of a detergent‐assisted cartridge precipitation workflow for non‐targeted quantitative proteomics

Assessing the precision of a detergent‐assisted cartridge precipitation workflow for non‐targeted quantitative proteomics

Amber‐codon suppression for spatial localization and in vivo photoaffinity capture of the interactome of the Pseudomonas aeruginosa rare lipoprotein A lytic transglycosylase

Lytic transglycosylase Slt of Pseudomonas aeruginosa as a periplasmic hub protein

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