Nov 07, 2025

IN PROGESS: Proteomics Workflow

This  protocol  is a draft, published without a DOI.
  • Johnson Truong1
  • 1CZ Biohub
Johnson Truong
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Protocol Citation: Johnson Truong 2025. IN PROGESS: Proteomics Workflow. protocols.io https://dx.doi.org/
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: In development
We are still developing and optimizing this protocol
Created: February 10, 2025
Last Modified: November 07, 2025
Protocol  Integer ID: 119901
Keywords: shotgun proteomics protocol for sample preparation, working shotgun proteomics protocol, proteomics workflow, progess, assaymap bravo, sample preparation, trap micro
Abstract
This is intended as a working shotgun proteomics protocol for sample preparation using S-Trap Micro followed by subsequent clean-up by Stage Tipping/AssayMap Bravo
Materials
Equipment:
BioTek Synergy Neo2

Materials:
Pierce BCA Protein Assay Kit w/ Standards: Thermo Scientific, A55864
Pierce Iodoacetamide (IAA), no-weigh format, 30 x 9.3 mg: Thermo Scientific, A39271
Pierce DTT, no-weigh format, 48 x 7.7 mg: Thermo Scientific, A39255
1M Triethylammounium Bicarbonate (TEAB): Thermo Scientific, 90114
S-Trap Micros (100 ug capacity), 80 traps: Protifi, C02-micro-80
Pierce Trypsin Protease, 5 x 100 ug: 90058
rLys-C mass spec grade, 15 ug: Promega, V1671
Protein Normalization / BCA Reaction
If samples are given as lysed cells: ensure samples are thoroughly thawed. Samples with 5% SDS should be allowed to room temperature as to allow it to re-solubilize in sample.

If SDS-present samples still appear cloudy, running it on a thermomixer at 1200 rpm and 32C should assist with resuspending SDS.
Calculate rough estimation of sample's protein concentration. This can be calculated by understanding cell type and its yield of protein, and then confluency of plate size.

Note
BCA Standard Curve Range is from 0.025 - 1.50 ug/uL for 7 levels. A blank row is also included.

Prepare samples to fit within BCA standard range.

If sample protein concentration is unclear, additional inputs can be performed.
Using a UV-compatible 96-well microplate:

Aliquot 5uL of BSA standard curve in triplicate in rows A1-A3 down to H1-H3. Row H1-H3 being blank (LCMS Water).

Aliquot 5uL of sample in triplicate. The sample position follow the same scheme top left to bottom right, starting from A4-A6.

For aliquoting, pipette the sample into the bottom corner of the well as to allow the BCA reagent to mix properly.

Additional sample inputs may be performed by 2.5uL or 1.25uL. Sample aliquots may also be scaled down if working with limit sample volume.


BioTek Gen5 microplate reader: BCA assay plate map

Using Pierce‱ BCA Protein Assay Kit: create the BCA reagent by mixing 50:1 of Reagent A:Reagent B. Calculate appropriate volume of reagent to create with a slight overage to dispense.

Add 200uL of resulting reagent into each sample well. A trough and multichannel pipette may be used during this step.
On BioTek Gen5 spectrophotometer, open Gen5 Software. Select "Piece BCA Assay.prt". Double-check protocol's plate map.

The protocol will shake for 5 minutes, incubate the plate at 37C for 30 minutes, then read the plate at 562 nm.
Save and export .csv output. Review R2 value of BSA curve. Review file for %CVs of samples.

Depending on input of samples, apply a dilution factor e.g. 2.5 uL sample input with a 5 uL standard input has a 2x factor in concentration.
Sample Preparation - Reduction/Alkylation/Acidification
Normalize sample concentration using BCA results and with appropriate sample buffer.

Target concentrations are typically the lowest sample concentration within the plate.

Note
S-Trap Micro loads between 1-100 ug.

Reduction: Aliquot DTT in LCMS Water to a concentration of 1mM in sample. Mix and incubate on thermomixer at RT for 1200 rpm/20 minutes.

Prepare a vial of lyophilized DTT (7.7 mg) in LCMS H2O to a concentration of 24mM. Vial can be prepared to concentration with 2mL of LCMS H2O. Ensure all DTT is dissolved.
Alkylation: Aliquot IAA in LCMS water to a concentration of 5mM in sample. Mix and incubate on thermomixer at RT for 1200rpm/20 minutes. Ensure to cover samples from light.

Prepare a vial of IAA in LCMS H2O to a concentration of 125mM. Vial can be prepared to concentration with 400uL of LCMS H2O. Ensure all IAA is dissolved. Protect from light.
Acidification: Aliquot 27.5% phosphoric acid to a concentration of 2.5% phosphoric acid in sample. Check sample for pH <1, add more phosphoric acid if not.

Prepare a vial of 27.5% phosphoric acid in LCMS H2O.
AB
Volume
Initial Sample (1-100 ug)23 uL
DTT (24 mM)1 uL
IAA (125 mM)1 uL
Phosphoric Acid (27.5%)2.5 uL
Wash/Bind Buffer165 uL
Total Volume 192.5 uL
Reagent addition table. Ratios may be adjusted accordingly
S-Trap - Digestion
From calculated final volume, add 6x amount binding/wash buffer (100 mM TEAB in 90% MeOH, adjusted ~7.5 pH with pure phosphoric acid) to sample. Vortex well and do not centrifuge. Samples may appear colloidal or cloudy.
Trap Protein. Transfer in 75 uL increments of sample to S-Trap atop a waste-flow container (1.7 mL or 2.0 mL tube). Spin at 4000g/30s. Rotate tubes 180 degrees between each addition and centrifugation to allow even flow through. Repeat as necessary until all sample has been captured and ensure all sample has flowed through by visually inspecting. If working in a centrifuge block: 2000g/30s.

75uL was determined to fit within the narrow stem of the S-Trap.
Clean Protein: Add 150 uL binding/washing buffer (100mM TEAB in 90% MeOH) to wash samples 4x.

Centrifuge at 4000g/30sec and discard flow as necessary. Centrifuge blocks: 2000g/30s.
Centrifuge S-Traps one last time to fully remove binding/wash buffer at 4000g/1 min or 2000g/30s on the centrifuge block.
Transfer S-Traps with loaded protein to clean 1.7 mL sample tube for digestion.
Digestion: Prepare digestion buffer with at least 1 ug protease/20 uL digestion buffer per sample in 50 mM TEAB buffer. Digestion buffer can prepared with trypsin or trypsin/Lys-C combination.

1) 1:25 ratio trypsin
2) 1:10 Lys-C and trypsin

A minimum of 1ug protease must be added onto S-Trap to ensure efficient digestion.
Aliquot 20uL of digestion buffer with protease onto trapped proteins. Ensure there is no air bubble on top of trap. S-Trap may be gently flicked to remove air bubbles of or spun down rapidly. Re-load any flow-through if the latter is performed.
Once all samples are loaded, transfer to a thermomixer that accept vials. Loosen the cap; do not tighten, as a vacuum may form and sample will be drawn through the trap.
Incubate at 47C for 1-2 hours. Do not shake, the digestion buffer may elute off the trap.
Elution 1: Add 40 uL of 50mM TEAB pH 8.5 directly on top of S-Trap digestion buffer. Centrifuge 4000g/1 minute or 2000g/30s for centrifuge block.
Elution 2:Add 40uL 0.2% FA. Centrifuge 4000g/1 minute or 2000g/30s for centrifuge block.
Elution 3: Add 40uL 50% ACN. Centrifuge 4000g/1 minute or 2000g/30s for centrifuge block.
Flash freeze with liquid nitrogen and speed vac down for ~1 hour
Reconstitute in 100 uL 1% TFA in Water and proceed to peptide cleanup.
Peptide Cleanup - StageTips
Employ a StageTip for each sample to be cleaned up.

For higher throughput (samples > 12), the tips can be used through the staging blocks.
[INSERT PHOTO]

For lower throughput (samples <12), the tips can be used through the tip centrifuge. The tip centrifuge spins at a higher speed which can be visibly confirmed if solvent has eluted through. Ensure the solvent waste bottle is not full.
[INSERT PHOTO]


Note
For deeper proteome coverage, a small-scale fractionation may be performed by increasing hydrophobicity and pH.

This is done by replacing the elution step in Step 35 with 3 additional elution reagents and steps. Theoretically this step will fractionate the sample into 3 equal concentrations.


Organize all necessary reagents in order of usage for StageTipping. Have enough of each solution for 100 uL/sample.
ACTIVATE: Add 100 uL of Methanol to StageTips. Centrifuge at 500g/5 minutes. Clean out eluant.

Methanol may take longer to elute.
CONDITION: Add 100 uL of 80% ACN/0.1% TFA. Centrifuge at 500g/3 minutes. Clean out eluant.
EQUILIBRATE: Add 100 uL of 0.2% TFA in Water. Centrifuge at 500g/3 minutes. Clean out eluant.
LOAD: Load sample onto StageTips. Centrifuge at 300g/5 minutes or longer if needed.

Label tips as for better organization. Avoid any precipitated pellet. Flow-through can be collected if desired. If not, clean out eluant.
WASH: Add 100 uL of 99% IPA + 1% TFA (0.1% TFA in final solution). Centrifuge at 500g/5 minutes. Clean out eluant.

IPA may take longer to elute.
WASH: PERFORM TWICE: Add 100 uL of 0.2% TFA in Water. Centrifuge at 500g/3 minutes. Clean out eluant.
WASH: Add 100 uL of 0.1% FA in Water. Centrifuge at 500g/3 minutes. Clean out eluant.
ELUTE: Add 100 uL of 60% ACN, 0.5% Ammonia and collect into 3x8 tubes or 8-strip tubes. Ensure tips are properly lined into collection tubes. Centrifuge at 300g/5 minutes.

Follow note below for small-scale fractionation; if not, continue to next step.


Note
Small-Scale Fractionation:

ELUTION 1: Add 20 uL. Centrifuge at 300 g/3 minutes. Move tips to next appropriate collection vial.
200 uL 500 mM ammonium formate
400 uL ACN
400 uL H2O
5 uL formic acid
ELUTION 2: Add 20 uL. Centrifuge at 300 g/3 minutes. Move tips to next appropriate collection vial.
300 uL 500 mM ammonium formate
600 uL ACN
100 uL H2O
5 uL formic acid
ELUTION 3: Add 30 uL. Centrifuge at 300 g/3 minutes.
800 uL ACN
200 uL of 5% ammonium hydroxide (160 uL of water + 40 uL of 25% ammonium hydroxide)


Flash freeze with liquid nitrogen. Centrivap for minimum 1 hour.
Reconstitute in MS loading buffer (2% ACN/0.1% TFA).
Protocol references