Feb 25, 2026
Preparation of tryptic peptides from cell lysates for quantitative proteomics for Karlodinium veneficum PLY720, Kryptoperidinium triquetrum CCAP 1116/3, Nitzschia laevis UTEX_B_2047 and Symbiodinium sp. - reduction, alkylation, digestion and desalting
- Will Lewis1,
- Ross F. Waller2
- 1Department of Biochemistry, University of Cambridge;
- 2University of Cambridge
Protocol Citation: Will Lewis, Ross F. Waller 2026. Preparation of tryptic peptides from cell lysates for quantitative proteomics for Karlodinium veneficum PLY720, Kryptoperidinium triquetrum CCAP 1116/3, Nitzschia laevis UTEX_B_2047 and Symbiodinium sp. - reduction, alkylation, digestion and desalting. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv55d9dv1b/v1
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: February 04, 2026
Last Modified: February 25, 2026
Protocol Integer ID: 242605
Keywords: preparation of tryptic peptide, tryptic peptide, cell lysates for quantitative proteomic, quantitative proteomic, kryptoperidinium triquetrum ccap, karlodinium veneficum ply720, symbiodinium sp, cell lysate
Funders Acknowledgements:
Gordon and Betty Moore Foundation
Grant ID: https://doi.org/10.37807/GBMF11532
Abstract
This method is for the preparation of tryptic peptides from cell lysates for quantitative proteomics for Karlodinium veneficum PLY720, Kryptoperidinium triquetrum CCAP 1116/3, Nitzschia laevis UTEX_B_2047 and Symbiodinium sp. - reduction, alkylation, digestion and desalting.
Guidelines
This protocol requires multiple samples to be processed in parallel. Often volumes of solutions need to be recalculated and adjusted based on changes made in earlier steps in the protocol. The easiest way to keep track of and calculate dilutions and volumes is by using a spreadsheet.
Materials
Equipment:
Visiprep SPE Vacuum Manifold, PTFE Free - standard, 24-port model
Vacuum pump
Sep-Pak tC18 desalting columns (Waters, P/N WAT054960)
Troubleshooting
Quantitation of protein concentration in cell lysates
Make a 40 µL 1 in 20 dilution of each cell lysate sample obtained using the protocol "Cell lysis for preparation of protein extracts for quantitative proteomics" in 1M TEAB.
For these pigmented algal samples, dilution is important as the concentrated pigments will cause the quantification to be overestimated.
Perform BCA assay with three technical replicates for each sample using the Pierce BCA Protein Assay Kits
Sample Dilution
To make pipetting easier, so that the same volume can be used for each sample throughout this protocol, diluting all samples to the same concentration as the sample with the lowest concentration is recommended. Use 8M Urea and 0.1 M TEAB in HPLC-grade water as diluent.
166 µg total protein quantity is required for each sample.
First calculate the volume of each sample required for 166 µg and transfer an aliquot of this volume to a 2 mL tube, using this equation:
Volume of undiluted sample aliquot (µL) = 166 / concentration measured by BCA assay (µg/µL)
Determine the volume of 8M Urea and 0.1 M TEAB in HPLC-grade water diluent to add to the cell lysate protein extract aliquot in the tube for each sample using this equation for each sample:
Volume of diluent required = volume of the largest aliquot for any sample in the current batch (µL) - the volume of the present sample (µL)
Add this volume to the aliquots. This should mean that the sample with that had the lowest concentration will require no further dilution and all samples will now be the same volume as this sample.
Reduction
In a fume hood prepare 100 mM stock of DTT in HPLC-grade water by dissolving 154 mg in 10 mL HPLC-grade water.
Aliquot in 0.5 mL volumes and store these at -20 °C for future use.
Add DTT to each sample to a target concentration of 5 mM. Incubate at 37 °C for 30 minutes.
Alkylation
Iodoacetamide stock should be prepared fresh before use. Do not save stocks for future use as they are likely to degrade. Protect from light and prepare the solution in an opaque or amber tube.
In a fume hood prepare a 375 mM stock of iodoacetamide (IAA) by dissolving 104 mg IAA in 1.5 mL HPLC-grade water.
Add IAA to each sample to a target concentration of 15 mM. Incubate in the dark at room temperature for 30 minutes.
First Trypsin/Lys-C digestion
Calculate the number of 20 µg aliquots of lyophilised Trypsin/Lys-C (Promega) so that there is sufficient quantity to add a minimum of 6.64 µg to each sample.
Reconstitute by adding 21 µl of 0.1 M TEAB to each 20µg aliquot.
To each sample add a 0.04 volume of reconstituted Trypsin/Lys-C (1:25 dilution).
Incubate at 37 °C in a shaker incubator with shaking at 200 rpm for 4 hours.
For each sample, add 8.1 parts 0.1 M TEAB to 1 volume of sample. This step dilutes the 8M urea present in the sample buffer, activating the trypsin.
Incubate at 37 °C in a shaker incubator with shaking at 200 rpm overnight (approximately 17 hours).
Second Trypsin/Lys-C digestion
Reconstitute the same amount of lyophilised Trypsin/Lys-C (Promega) used in the first digestion, and add the same volume containing 6.64 µg to each sample.
Incubate at 37 °C in a shaker incubator with shaking at 200 rpm overnight.
Termination of digestion
Terminate digestion by adding trifluoroacetic acid (TFA) to a final concentration of 0.5–1%
Remove particulate material by centrifuging at 15,000 × g for 10 minutes and retaining the supernatant for each sample.
Buffer exchange
The presence of TEAB in the samples can interfere with peptide binding to Sep-Pak columns during downsteam desalting steps, and therefore exchanging the buffer for 0.1% trifluoroacetic acid (TFA) and 2% acetonitrile (ACN) in HPLC-grade water provides much greater recovery of purified peptides.
Dry the samples using a Speed-Vac vacuum concentrator such as the Thermo Scientific Savant SpeedVac SPD10302.
Resuspend in 400 µL 0.1% trifluoroacetic acid (TFA) and 2% acetonitrile (ACN) in HPLC-grade water.
Confirm pH < 2.5 by pipetting 2 µL aliquots onto suitable pH indicator strips for each sample.
Samples can be stored at -20 °C. The downstream desalting process will take 1-2 hours.
Desalting
First prepare required buffers
For each of these buffers, prepare sufficient volume for the total number of samples being processed plus two (n + 2), to account for pipetting error.
1) Activation Buffer
1 mL Methanol, per sample
2) Conditioning Buffer
2 mL ACN, per sample
3) Equilibration and Washing Buffer
0.1% TFA: 6 µL TFA in 6 mL HPLC-grade water, per sample
4) Elution Buffer
1.2 µL TFA, 0.6 mL HPLC-grade water and 0.6 mL ACN, per sample, and mix well.
| A | B | C | D | E | |
| Stock solutions | Per sample | 15 (+2) samples | 20 (+2) samples | 25 (+2) samples | |
| Activation | 1 mL MeOH | 17 mL MeOH | 22 mL MeOH | 27 mL MeOH | |
| Conditioning | 2 mL ACN | 34 mL ACN | 44 mL ACN | 54 mL ACN | |
| Equilibration and washing | 6 µL TFA 6 mL H2O | 102 µL TFA 102 mL H2O | 132 µL TFA 132 mL H2O | 162 µL TFA 162 mL H2O | |
| Elution | 0.6 µL TFA 0.3 mL ACN 0.3 mL H2O | 10.2 µL TFA 5.1 mL ACN 5.1 mL H2O | 13.2 µL TFA 6.6 mL ACN 6.6 mL H2O | 32.4 µL TFA 16.2 mL ACN 16.2 mL H2O |
Table of pre-calculated volumes for common sample batch sizes
Use of a repeater pipette is recommended for loading buffer to the columns in the following steps.
For each sample being processed, insert a Sep-Pak tC18 desalting column (Waters, P/N WAT054960) into a luer valve adapter in the Visiprep SPE Vacuum Manifold and load a 15 mL tube at each corresponding position in the collection tube rack.
Activation
Pass 1 mL MeOH through each column using the vacuum.
Conditioning
Pass 2 x 1 mL ACN through each column using the vacuum.
Equilibration
Pass 2 x 1mL 0.1% TFA in HPLC-grade water through each column using the vacuum.
Turn off vacuum and release pressure from the manifold via the vacuum pressure release valve on the front of the manifold. The samples must be passed through the column slowly to maximise binding.
Optional: If you would like to maximise recovery by passing each sample through the column twice, or if you are concerned about losing material and would like to therefore save the sample flow-through incase the sample fails to effectively bind to the column, replace the 15 mL collection tubes with clean, sterile 15 mL tubes.
If the TEAB-containing sample buffer was exchanged for 0.1% trifluoroacetic acid (TFA) and 2% acetonitrile (ACN) in the upstream steps of this protocol, this step is unlikely to be necessary.
Sample Loading
Ensuring all vacuum pressure has been released, load each sample into a equilibrated Sep-Pak column.
With the vacuum pressure release valve on the front of the manifold still open, turn back on the vacuum pump. Slowly partially close the vacuum pressure release valve so the pressure starts gradually increasing. As soon as the samples being moving through the columns, release the pressure again to pause this process. Take 30 seconds pause before gradually closing the vacuum pressure valve again. This is the easiest way to control a slow rate while passing the samples through the columns. Ideally, for maximum retention, the samples should slowly and incrementally be passed through the columns over the course of around 5 minutes.
Optional: If saving the sample flow-through, remove the 15 mL tubes at this point, ensure that they are labelled with the words "flow-through" and the corresponding sample and replicate ID, and store at -20 °C.
Washing
Pass 4 x 1mL 0.1% TFA in HPLC-grade water through each column using the vacuum.
Turn off vacuum and release pressure from the manifold via the vacuum pressure release valve on the front of the manifold.
Replace 15 mL tubes with clean, sterile 15 mL tubes. Eluting directly into 1.5 mL tubes risks sample loss, since the eluate can bubble as it passes through the outlet, which can cause it to spill over the narrow openings of 1.5 mL tubes.
Upon writing this protocol in a format for protocols.io it has occured to me that some metal luer nozzles were included with the Visiprep manifold. I have never known what these were for, but it could be that they are intended as outlet extenders, allowing outlets to extend deeper into 1.5 mL tubes, and thereby reducing the chance of sample loss due to the eluate bubbling over the opening of the tube. However, I have never tested whether this. If it is the case, then this might be substantially easier and create less plastic waste than collecting the eluted samples in 15 mL tubes, and then transferring to 1.5 mL tubes.
Elution 1
Apply a 250 µL volume of 50% ACN and 0.1% TFA in HPLC-grade water to each column and allow to stand for 30 seconds before passing through the column using the vacuum into the collection tube.
Elution 2
Apply another 250 µL volume of 50% ACN and 0.1% TFA in HPLC-grade water to each column and allow to stand for 30 seconds before passing through the column using the vacuum, into the same collection tube as elution 1.
Fluorometric peptide quantitation assay
Measure the peptide concentration of each sample using the Pierce Quantitative Peptide Assay kit.
Measure three 10 µL volumes as technical replicates for each purified peptide sample.
Diluting the sample at this stage is not recommended as the concentration could fall below the quantitation limit for this assay.
Dilute and dry samples
Using the peptide assay results, calculate the volume of each sample required to obtain 20 μg of purified peptide using the formula:
Volume (μL) = (20 μg) / [peptide concentration (μg/μL)]
Transfer the calculated volume of each sample into a clean, labeled tube. As these are the tubes that will be submmited to the proteomics mass spectrometry facility, each tube should be clearly marked with:
- Sample ID
- Replicate ID
- Date
- Peptide amount (20 μg)
Dry samples using the Speed-Vac and either store at -20 °C or submit immediately to the proteomics mass spectrometry facility.