Sep 04, 2025
Version 2
PSMtag - Anhydrous Labeling Protocol (50 µg Labeling) V2 V.2
- Mike Agius1,
- ssipe 1,
- Sarah Sipe1
- 1Parallel Squared Technology Institute
Protocol Citation: Mike Agius, ssipe , Sarah Sipe 2025. PSMtag - Anhydrous Labeling Protocol (50 µg Labeling) V2. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3w5yzv25/v2Version created by Sarah Sipe
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 03, 2025
Last Modified: September 04, 2025
Protocol Integer ID: 226375
Keywords: Mass Tags, Anhydrous Labeling, Proteomics, Peptide Labels, PSMtag, PTI, labeling peptide, peptides with psmtag, 50ug peptide sample, peptide to tag ratio, anhydrous labeling protocol, peptide, use of psmtag, general protocol for labeling, labeling, 75ug of psmtag, labeling input, psmtag, allowing robust labeling efficiency, robust labeling efficiency, tag ratio, µg labeling
Abstract
A water-free (anhydrous) method labeling peptides with PSMtag. Similar methods enable the use of PSMtag as low as 1 to 1 (wt/wt) peptide to tag ratios. What follows is a general protocol for labeling of an 50ug peptide sample using 75ug of PSMtag (1.5X wt/wt), allowing robust labeling efficiency of >98%. Labeling input can be scaled accordingly.
Guidelines
Key points while scaling up or down:
- Maintain Peptide concentrations > 1 mg/mL
- Maintain PSMtag concentrations > 28.4 mM
- DIPEA is calculated to be a 30eq peptide molarity
- All uses of "DMSO" refer to using fresh, anhydrous DMSO
Key points while scaling down:
- This protocol has been demonstrated to easily scale down to 8µg peptide input
- Recommendations for scaling down further to be determined soon
Materials
1. PSMtag – 56.7 mM in DMSO (24 µg/µL).
2. N,N-Diisopropylethylamine (DIPEA) – 5740 M pure (Sigma cat: D125806)
3. Anhydrous DMSO (Sigma cat: 276855)
4. 1M Triethylammonium Bicarbonate, TEAB (Sigma cat: T7408)
5. 0.2 mL PCR tubes (Thermo Fisher cat: E0030124286)
6. Hydroxylamine, HA - 50% in water (Sigma cat: 8.14441)
Before start
Material Preparation:
- Use fresh, anhydrous DMSO to dissolve PSMtag and peptides, as traces of water can create undesirable hydrolysis products during the labeling reaction.
- Freeze PSMtag in aliquots at -80ºC to avoid repeated freeze-thaw cycles.
- Solubilizing desalted peptides in TEAB and re-drying helps basify the reaction if previously dried from a formic acid solution.
PSMtag Anhydrous Labeling Protocol (50µg of Digest)
3h 15m
An aliquot of 50 µg of desalted peptide digest is resuspended in 100 µL of 100 mM TEAB and dried to completeness in a 0.2 mL tube for 1 hr under vacuo using a speed-vac.
1h
Dissolve sample in 3.25 µL of a 436.6 mM DIPEA in DMSO (~30 eq).
Labeling by PSMtag is initiated by the addition of 3.25 µL of 56.7 mM PSMtag in DMSO (4 eq).
Pipette and Spin 0.2 mL tube to mix - ensuring all peptide sample is completely dissolved from the tube walls.
Labeling reaction is incubated at 25 ºC for 2 hrs.
2h
Reaction is quenched with 1 µL of a 1.8% HA solution in water (v/v) and incubated 25 ºC for 30 min.
15m
Sample is either stored at -20 ºC or further diluted for LC-MS/MS analysis.
Preparation for LC-MS/MS Analysis
Bulk PSMtagged peptides can be diluted directly after labeling for low-input analyses. Recommended dilution solvent is 30% acetonitrile, 2% formic acid, and 0.05% DDM (n-dodecyl β-D-maltoside). Final DMSO concentration should be <5%
If drying samples for storage, it is recommended to reconstitute first with organic solvent (i.e. acetonitrile or DMSO), then dilute with aqueous solutions to reach desired injection conditions (see step 8).
Note
It is NOT recommended to reconstitute in entirely aqueous solvent. At least 20% DMSO in water (or ACN) is necessary to solubilize samples.