Feb 11, 2025
Absolute Proteome Quantification of Extracellular Proteins
- Borja Ferrero Bordera1,2,
- Sandra Maaß3
- 1LMU Munich;
- 2University Greifswald;
- 3University of Greifswald
Protocol Citation: Borja Ferrero Bordera, Sandra Maaß 2025. Absolute Proteome Quantification of Extracellular Proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxwnyov8j/v1
Manuscript citation:
Ferrero-Bordera B, Bartel J, van Dijl JM, Becher D, Maaß S.2024.From the outer space to the inner cell: deconvoluting the complexity of Bacillus subtilis disulfide stress responses by redox state and absolute abundance quantification of extracellular, membrane, and cytosolic proteins. Microbiol Spectr12:e02616-23.https://doi.org/10.1128/spectrum.02616-23
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: December 20, 2024
Last Modified: February 11, 2025
Protocol Integer ID: 116532
Keywords: Proteomics, Mass-Spectrometry, Absolute Quantification, Bioinformatics, Python, Exoproteome, Extracellular proteins
Funders Acknowledgements:
People Programme (Marie Skłodowska-Curie Actions) of the European Union’s Horizon 2020 Programme
Grant ID: 813979
Abstract
To quantify secreted proteins in the culture medium, we described a simple and straightforward protocol for the absolute quantification of extracellular proteins in bacteria. We concentrated extracellular proteins, which are highly diluted in the medium, using StrataClean resin along with a set of standard proteins to determine the extent of the concentration step.
Materials
Equipment
- Low-binding tubes
- Centrifuge
- Overhead shaker
- SpeedVac centrifuge
Common Reagents & Buffers
- Tris-EDTA (TE) buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5)
- StrataClean resin (Agilent)
- 37 % HCl
- Loading buffer (4% (w/v) SDS, 20% (v/v) glycerol, 40 µg/mL bromophenol blue, 125 mM Tris-HCl pH 6.8 with 10% (v/v) β-mercaptoethanol)
- Porcine Trypsin
Protein Standards
- Anchor proteins (e.g. Universal Protein Standards -UPS2- from Sigma)
- Enrichment standards (described in this protocol)
Extracellular protein extraction
Extracellular protein extraction
1d 1h
1d 1h
Pellet by centrifuging at 4 °C an aliquot of the culture to be analysed. 8500 x g, 4°C, 00:10:00
10m
Recover the supernatant into a new tube.
Note
Low-binding tubes are recommended when preparing proteomics samples.
10m
Add the Enrichment Standards to the raw supernatant. Important! This step is crucial to quantify the total protein in the raw supernatant allowing to estimate the protein binding efficiency.
Standards were added based on the supernatant's protein concentration in a ratio of 1:35 (ng of standard mixture: ng of estimated sample protein). Below is the table of proteins used as standards in our paper.
| Protein | Organism | Accession | Supplier | Concentration (ng/µL) | Protein amount (fmol) | |
| alpha-Lactalbumin | Bos taurus (Bovine) | P00711 | Sigma Merck | 174.07 | 5000 | |
| Glycerol-3-Phosphate dehydrogenase | Oryctolagus cuniculus (Rabbit) | P46406 | Sigma Merck | 460.05 | 5000 | |
| Alcohol dehydrogenase | Saccharomyces cerevisiae (Yeast) | P00330 | Sigma Merck | 45.76 | 500 | |
| Soybean trypsin inhibitor | Glycine may (Soybean) | P01071 | Gibco | 24.87 | 500 | |
| Lisozyme | Gallus gallus (Chicken) | P00698 | Pierce | 17.78 | 500 | |
| Bovine serum albumin | Bos taurus (Bovine) | P02769 | Pierce | 8.60 | 50 | |
| Carbonic anhydrase | Bos taurus (Bovine) | Q1LZA1 | Sigma Merck | 3.61 | 50 |
10m
Add 20 µL HCl-primed StrataClean resin (Agilent; REF 400714) into the raw supernatant and incubate at 4 °C overnight with overhead shaking.800 rpm, 4°C, 16:00:00
Note
Other protocols (TCA/Acetone precipitation) can be used as they showed comparable efficiency to the one described here. Nonetheless, StrataClean beads reduce the usage of organic solvent
1d
Centrifuge at maximum speed at 4 °C and discard the supernatant to recover the resin with the bound proteins. Wash the resin with TE buffer and repeat the centrifugation step.
30m
Vacuum-dry the resin containing the bound protein.
30m
Protein Electroelution
Protein Electroelution
1h 40m
1h 40m
Resuspend the resin-bound protein in 20 µL loading buffer (consider the gel pocket size to adjust the final volumes, as it will be combined with the anchor proteins in the well).
Note
It is recommended to resuspend the dried resin in 2X the original amount to ease the pipetting into the gel as the resin is very viscous. For example, 20 µL initial resin can be divided into 2 eppis with 10 µL each, dried and resuspended in 20 µL loading buffer.
10m
Load the resin with the bound protein into the gel pocket. After sample loading, add the anchor proteins used for quantification (e.g. UPS2) into the gel pocket on top.
10m
Run the SDS-PAGE gel to the desired time. Lower times are recommended for samples with low complexity and/or highly diluted.
50m
Proceed with Coomassie staining to visualize the eluted protein in the gel lanes.
30m
In-Gel protein digestion & desalting
In-Gel protein digestion & desalting
1d 1h 5m
1d 1h 5m
Fractionate the gel lanes in squares of 2x2 mm
20m
Destain the gel with 200 mM ammonium bicarbonate buffer in 30% (vol/vol) acetonitrile with 00:15:00 incubations at 37 °C . Repeat until completely destained.
45m
Vacuum-dry the destained gel pieces and resuspend them in 2 µg/µL trypsin. Incubate overnight at 37 °C.
1d
To elute the digested peptides, resuspend the digested gel pieces in MilliQ-water and sonicate for 15 minutes. Centrifuge at maximum speed and transfer the supernatant to a low-binding eppi.
Vaccum-dry and resuspend in 0.1 % MS-grade acetic acid.
Proceed with desalting. We recommend ZipTip (Millipore) desalting because of the simplicity of the protocol. Nonetheless, other protocols like StageTipping can be performed.
Vaccum-dry and resuspend with Buffer A for MS measurement of the samples.