tmtpro 16-plex reagents  (Thermo Fisher)

Journal: bioRxiv

Article Title: Synergistic Arg-C Ultra and Lys-C Digestion for Quantitative Proteomics

doi: 10.1101/2025.07.15.664461

Figure Lengend Snippet: (a) Schematic overview of a standard Lys-C/Trypsin workflow. Lysates undergo protein cleanup using SP3 precipitation. Bead-bound proteins are initially digested overnight with Lys-C (20 ng/uL) in 2 M GuHCl, 10 mM EPPS (pH 8.5) at 24 °C. The following day, the GuHCl concentration is diluted to 0.5 M, and digestion proceeds overnight at 37 °C with an additional Lys-C (20 ng/uL) and Trypsin (10 ng/uL). For label-free experiments, samples are separately desalted via C18 cleanup and analyzed on the mass spectrometer. For multiplexed analysis samples are vacuum-evaporated, resuspended in 200 mM EPPS (pH 8.0), labeled with TMTpro, combined, and desalted. Created with BioRender.com. (b) Proteomics analysis of HeLa lysate via TMTproC and label-free DIA reveals cleavage efficiencies from the standard workflow, showing ∼5% of peptides where the first basic amino acid is lysine (K) containing a missed cleavage, and 30% of peptides where the first basic amino acid is arginine (R) containing a missed cleavage.

Article Snippet: For TMTpro labeling, peptides were incubated with TMTpro reagents (Thermo Fisher, A52045) at a 7.5:1 tag-to-peptide mass ratio for 2 hours at room temperature.

Techniques: Concentration Assay, Mass Spectrometry, Labeling

Journal: bioRxiv

Article Title: Synergistic Arg-C Ultra and Lys-C Digestion for Quantitative Proteomics

doi: 10.1101/2025.07.15.664461

Figure Lengend Snippet: Icons indicate experiment type: shotgun proteomics or UV-Vis. (a) Overview of nucleophilic (Nu) hydrolysis susceptibility of isobaric tags like TMTpro NHS-Ester. Common reducing agents (e.g., β-mercaptoethanol (BME), dithiothreitol (DTT)) can hydrolyze NHS- esters, potentially impairing TMTpro labeling efficiency. (b) UV-Vis absorbance spectra of intact and hydrolyzed TMTpro0 NHS-ester. Intact TMTpro0 weakly absorbs at 260 nm, while hydrolyzed strongly absorbs. Absorbance at 260 nm was converted to concentration using Beer’s Law (ε = 9700 M⁻¹cm⁻¹) . (c) Comparative assessment of NHS-ester hydrolysis by reducing agents BME, DTT, and TCEP. Without reducing agents, spontaneous hydrolysis occurs due to hydroxide ion interactions. DTT and BME (1 mM) rapidly induce extensive NHS-ester hydrolysis, whereas TCEP at the same concentration causes substantially less hydrolysis. (d) UV-Vis assay showing increased TMTpro NHS-ester hydrolysis with rising TCEP concentrations. (e) Effect of TCEP on TMTpro Labeling Efficiency. Shotgun proteomics analysis showing that HeLa peptides labeled with TMTpro0 in the presence of 1 mM TCEP achieve labeling efficiencies comparable to TCEP-free conditions, while 5 mM TCEP reduces labeling efficiency, increasing the fraction of unlabeled peptides by 23%. TMTpro0 was searched as a variable modification on lysine residues and peptide N-termini. Error bars represent ±1 standard deviation; n = 3 for all experiments. For proteomics experiments, each replicate was labeled independently. Shotgun proteomics data were acquired using a TMTpro-SPS-MS3 method.

Article Snippet: For TMTpro labeling, peptides were incubated with TMTpro reagents (Thermo Fisher, A52045) at a 7.5:1 tag-to-peptide mass ratio for 2 hours at room temperature.

Techniques: Labeling, Concentration Assay, Modification, Standard Deviation

Journal: bioRxiv

Article Title: Synergistic Arg-C Ultra and Lys-C Digestion for Quantitative Proteomics

doi: 10.1101/2025.07.15.664461

Figure Lengend Snippet: (a) Schematic of Lys-C/Arg-C Ultra digestion workflow compatible with multiplexed and label-free proteomics. Bead-bound proteins are initially digested with Lys-C (40 ng/uL) in 20 mM EPPS (pH 8.5) at 37 °C overnight. TCEP was then added to 1 mM, followed by Arg-C Ultra at a 1:200 enzyme-to-protein ratio, with a second overnight digestion performed at 24 °C. Samples were either analyzed label-free or labeled with a unique TMTpro tag. After C18 cleanup, samples were analyzed with TMTproC or label-free DIA. Created with BioRender.com. (b) The Lys-C/Arg-C Ultra workflow is highly reproducible. To evaluate reproducibility, a single precipitated HeLa lysate was divided into 6 identical aliquots and digested independently. This was done with both the Lys-C/Trypsin protocol and the Lys-C/Arg-C Ultra protocol. For comparison, we premixed the six TMTpro tags and labeled a single pool of HeLa peptides (from Lys-C/Arg-C Ultra digest) with this mixture (grey). Samples were analyzed by TMTproC (unfractionated). This distribution represents the experimental limit of reproducibility by eliminating variability due to sample preparation, thus revealing only the variability that arises from the data acquisition process, such as limited ion statistics. A stringent signal-to-noise cutoff of 852 (2+) and 2206 (3+) was used. (c) Lys-C/Arg-C Ultra digestion minimizes missed cleavages at arginine residues while maintaining high efficiency at lysine sites. When applied to DIA workflows on an Orbitrap Ascend with 200 ug loaded, missed cleavages were reduced from ∼30% to <1% for peptides where the first basic amino acid is arginine (R), while remaining <5% for peptides where the first basic amino acid is lysine (K), compared to Lys-C/Trypsin. Error bars represent ±1 standard deviation from n = 3 replicates (digested independently). (d) Lys-C/Arg-C Ultra digestion improves peptide and protein identifications for label-free DIA proteomics. From the same samples in (c), Lys-C/Arg-C Ultra digestion increased the number of unique, fully cleaved peptides by 23% and protein identifications by 6% compared to Lys-C/Trypsin digestion. (e) Lys-C/Arg-C Ultra digestion improves peptide and protein identifications for multiplexed proteomics. From the samples described in (b), Lys-C/Arg-C Ultra digestion increased the number of unique, fully cleaved peptides by 29% and protein identifications by 11% compared to Lys-C/Trypsin digestion. A sum signal-to-noise ratio cutoff of 136 (2+) and 353 (3+) was applied, corresponding to a 10% coefficient of variation (CV) from ion statistics , .

Article Snippet: For TMTpro labeling, peptides were incubated with TMTpro reagents (Thermo Fisher, A52045) at a 7.5:1 tag-to-peptide mass ratio for 2 hours at room temperature.

Techniques: Labeling, Comparison, Sample Prep, Standard Deviation