A Suspension-Trapping Protocol for Bottom-Up Proteomics Sample Preparation

Materials and reagents

Biological materials

1. Frozen rat cerebrospinal fluid sample

Note: In our work, 31 samples were used. This protocol has also been successfully conducted using human cerebrospinal fluid and plasma samples.

Reagents

1. Trizma base (Tris) (Sigma-Aldrich, catalog number: T1503-1KG)

2. Phosphoric acid (Honeywell, catalog number: 79606-500 mL)

3. Pierce^TM BCA Protein Assay kit with Dilution-Free^TM BSA protein standards (Thermo Fisher Scientific, catalog number: A55865).

4. High-Select^TM Top14 abundant protein depletion resin (Thermo Fisher Scientific, catalog number: A36366)

5. S-Trap^TM micro column (≤100 μg; PROTIFI, CO₂-micro-10, CO₂-micro-40, or CO₂-micro-40, depending on numbers of samples processed)

6. Dithioreitol (DTT) (Sigma-Aldrich, catalog number: D9779-1G)

7. Iodoacetamide (IAA) (Sigma-Aldrich, catalog number: I1149-5G)

8. Trypsin w/ CaCl₂ (TPCK-treated, 500 μg), 10 pack (SCIEX, catalog number: 4445250)

9. Milli-Q water

10. Formic acid (Sigma-Aldrich, catalog number: 695076-100ML)

11. Acetonitrile (ACN) CHROMASOLV LC-MS (Honeywell, catalog number: 34967-4X4L)

12. Sodium deoxycholate (DOC) (Sigma-Aldrich, catalog number: 30970-25G)

13. Ammonium bicarbonate (ABC) (Sigma-Aldrich, catalog number: A6141-25G)

14. Triethylammonium bicarbonate buffer (TEAB) (Sigma-Aldrich, catalog number: T7408-100 mL)

15. Dithiothreitol (DTT) solution, 1 M (ThermoFisher Scientific, catalog number: P2325)

16. PBS solution, pH 7.4 (1×, 10 mM) (Gibco^TM, ThermoFisher Scientific, catalog number: 10010023)

Note: Trypsin platinum, mass spectrometry grade (100 μg, Promega, catalog number: VA900) is recommended for samples such as cerebrospinal fluid. For tissue or cell lysate samples, suitable alternatives include trypsin platinum, mass spectrometry grade (100 μg, Promega, catalog number: V5280) or sequencing-grade modified trypsin (lyophilized, 5 × 20 μg, Promega, catalog number: V5111)

Solutions

1. Lysis buffer (see Recipes)

2. 50 mM ammonium bicarbonate (pH ~8) containing 3% (w/v) DOC (see Recipes)

3. Aqueous buffer (see Recipes)

4. S-trap binding buffer, pH 7.1 (see Recipes)

5. Buffer A (see Recipes)

6. Buffer B (see Recipes)

Recipes

1. Lysis buffer

Note: The composition of the lysis buffer may vary in SDS concentration, typically 2% or 10%. SDS functions primarily to denature proteins, prevent aggregation, and maintain protein solubility. Higher concentrations (e.g., 10%) can be helpful for challenging samples such as skin tissue or membrane protein complexes; however, they also increase the complexity of downstream cleanup. In this protocol, we used 2% SDS, which was effective for cerebrospinal fluid (CSF) samples. A detailed recipe for high-concentration SDS and other buffer formulations is available upon request.

2. 50 mM ammonium bicarbonate (pH ~8) containing 3% (w/v) DOC

3. Aqueous buffer

4. S-trap binding buffer, pH 7.1

Note: Rinse glassware with 100% methanol before preparing S-Trap binding buffer.

5. Buffer A

6. Buffer B

Laboratory supplies

1. Pipette tips (TipOne 10, 200, 1,000 μL) (USA Scientific, catalog numbers: 1111-3800, 1110-1800, 1111-2821)

2. Pipettes (ErgoOne single-channel pipette 2.5, 10, 200, 1,000 μL) (USA Scientific, catalog numbers: 7100-0125, 7100-0510, 7100-2200, 7110-1000)

3. Pour boat weigh dish 2-1/4”l ×1-3/4”w × 5/16”d, 20 mL cap (Wilkem Scientific, catalog number: 10177901)

4. Pipette tip gel loading 0.57 mm O.D. 200 μL round non-sterile (Wilkem Scientific, catalog number: LABB13790)

5. Microcentrifuge tube 0.5 mL non-sterile (Cell Treat, Wilkem Scientific, catalog number: 72316004)

6. Microcentrifuge tube 1.5 mL non-sterile (Cell Treat, Wilkem Scientific, catalog number: 229441)

7. Microcentrifuge tube 1.7 mL non-sterile (Cell Treat, Wilkem Scientific, catalog number: 229441)

8. S-trap^TM 96-well mini plate (PROTIFI, catalog number: P002-96MINI)

9. 96-well sample collection plate (Waters, catalog number: 186002842)

10. Sealing mat for 96-well plate, polypropylene, round well (Waters, catalog number: 186002483)

11. 96-well filter plate, 0.2 µm, 1 mL (AcroPrep Advance, Cytiva, catalog number: 8686)

Note: Low protein-binding microcentrifuge tubes (e.g., SARSTEDT, catalog number: 72.706.600) are recommended to minimize peptide adsorption and improve recovery, especially for protein-limited samples such as cerebrospinal fluid.

Equipment

1. Eppendorf Vacufuge plus 5305 (Eppendorf AG, catalog number: 5305FQ525373)

2. Savant SpeedVac SPD210 vacuum concentrator (Thermo Scientific, catalog number: SPD210-P1)

3. Nanodrop One (ThermoFisher, catalog number: ND-ONE-W)

4. Eppendorf Thermomixer F1.5 (Eppendorf AG, model: 5384)

5. Grant-bio PMS-1000i (Grant Instrument Ltd., model: V.2GW.001)

6. Vortex MAXI MIX 1 (Thermo Scientific, Model: M16715)

7. Barnstead Thermolyne LABQUAKE Shaker Rotisserie (catalog number: 3625485)

8. Tecan (Resolvex) A200 Positive Pressure Manifold System

9. Thermo Fisher Orbitrap Ascend Tribrid Mass Spectrometer

Software and datasets

1. Spectronaut^® (Version 18.0, Biognosys AG)

3. Rat UniProt FASTA database (UP000002494_10116_Rat_Reference_20240602.fasta) and Universal Contaminant Protein FASTA (created 06.01.22, used 06.03.25) were used for protein identification and to generate the library for DirectDIA analysis

Procedure

A. Preparation of chemicals and reagents (tube format)

1. Prepare lysis buffer as described in Recipes.

Note: For each sample, mix 125 μL of 10% SDS with 500 μL of 100 mM Tris.

2. Prepare DTT working solution (160 mM): Dissolve 15.4 mg of DTT in 1 mL of Milli-Q water.

3. Prepare IAA solution: Add 36.98 mg of IAA (4 °C) to 1 mL of Milli-Q water.

4. Prepare phosphoric acid solution (12%): Mix 420 μL of 85% H₃PO₄ stock with 2,580 μL of Milli-Q water.

5. Prepare S-trap binding buffer as described in Recipes.

6. Prepare trypsin stock: Resuspend trypsin in 50 mM Tris, pH 8.5, at a concentration of 1 mg/mL.

7. Prepare elution buffer 1 (50 mM TEAB): Mix 100 mM TEAB with the same volume of Milli-Q water.

8. Prepare elution buffer 2 (0.2% formic acid).

9. Prepare elution buffer 3 (50% acetonitrile in water with 0.2% formic acid).

10. Prepare aqueous buffer as described in Recipes.

B. Depletion of Top 14 high-abundance proteins

B1. Top 14 protein depletion (tube format)

1. Equilibrate the depletion spin column to room temperature for at least 5 min.

2. Remove the screw cap from the column.

3. Add up to 80 μL of the CSF sample directly to the resin slurry in the column.

4. Securely cap the column and invert several times to homogenize the resin.

5. Incubate for 10 min at room temperature with gentle end-over-end mixing. Alternatively, vortex gently every 2–3 min. Incubation time can be extended to 20 or 30 min to increase depletion efficiency.

6. After incubation, remove the bottom closure by snapping it off.

7. Loosen the top cap and place the column into a 2 mL collection tube. Centrifuge at 1,000× g for 2 min.

8. Following centrifugation, discard the used column containing the resin and retain the filtrate, which is the sample depleted of high-abundance proteins.

9. Resuspend the sample in 10 mM PBS (pH 7.4, 1×) containing 0.02% sodium azide, pH 7.4. Proceed with downstream processing or store the sample at -20 °C for later use.

10. Transfer the collection tube to an Eppendorf Vacufuge. Set up 4 h and 30 °C on the V-AQ mode to dry the sample. Alternatively, the Centrivap step may be done at room temperature to avoid sample loss.

Note: High-Select^TM Top14 Abundant Protein Depletion Resin has a different catalog number. A36369 and A36370: Mini spin columns with a 200 μL antibody resin bed, optimized to bind up to 600 μg of serum or plasma; A36371 and A36372: Midi spin columns with a 500 μL antibody resin bed, optimized to bind up to 6,000 μg of serum or plasma. After step B1.10, check if there is any liquid remaining in the collection tube. If so, extend the centrifugation time as needed. Protein concentration can be estimated using a NanoDrop following step B1.10. To optimize the current procedure, we avoid concentrating samples with heat, as elevated temperatures may cause artifacts and sample loss. Instead, turn off the temperature setting, use a chilled Centrivap, or employ a Labconco lyophilizer.

B2. Top 14 protein depletion (plate format)

1. Add 200 μL of Pierce High-Select Top 14 abundant protein depletion resin to a 0.8 mL 96-well storage plate.

2. Add 40 μL of CSF sample to each well.

3. Seal the plate with a round-top sealing mat.

4. Holding the sealing mat and storage plate firmly, vortex moderately for 30 s.

5. Ensure the mat is properly sealed and then carefully invert the plate several times to mix the solution thoroughly. Return the plate to the upright position and then vortex for 5 min (Figure 1).

6. Secure the plate on a plate rotator and rotate for 20 min.

7. Pulse centrifuge the plate for 10–15 s (with the mat still on) to push the resin to the bottom of the wells.

8. Remove the sealing mat.

9. Mix the contents using a multichannel pipette and transfer to an Acro 8686 96-well filter plate positioned on top of a 2 mL square-well collection plate.

10. Add 200 μL of PBS (10 mM, pH 7.4, 1×) to the storage wells, mix by pipetting, and transfer the remaining resin to the filter plate.

11. Centrifuge at 8,000× g for 5 min to push the liquid through the filter into the collection plate.

12. Place the collection plate on a SpeedVac concentrator to dry.

C. Proteolytic digestion using S-trap

C1. Trypsin digestion (tube format)

1. Resuspend samples in 175 μL of 1× lysis buffer in a collection tube (see Recipes).

Note: A normalized amount of protein (e.g., 25 μg) could be combined with an equal volume of 2× lysis buffer to achieve a final concentration of 2% SDS in either 50 mM TEAB or 50 mM Tris. The total protein input is maintained within the recommended digestion capacity of the S-Trap columns (10–100 μg total protein), corresponding to an effective working concentration range of approximately 0.2–1 μg/μL for starting materials (e.g., plasma, cerebrospinal fluid, and plasma samples).

2. Add 25 μL of 160 mM DTT into the collection tube for a final concentration of 20 mM. Mix by pipetting while adding (the 160 mM DTT working solution can be prepared from DTT powder or by dilution from a 1 M DTT stock solution).

3. Cover the collection tube and heat at 95 °C for 15 min on the thermomixer with shaking at 300 rpm (approximately 0.3× g, 3 mm orbit). For laboratories without a thermomixer, static incubation at 95 °C with gentle mixing of the tube every 5 min can help achieve even heating and prevent localized overheating.

Note: Incubation at 70 °C for 30 min has proven effective. At sufficient concentrations, such as 10% SDS, denaturing proteins and larger proteins such as immunoglobulins are removed, leaving primarily lower-molecular-weight proteins, which can typically be denatured at 37 °C for 1 h or 55 °C for 45 min with shaking. For difficult-to-denature proteins or membrane-bound fractions, incubation at 70 °C for 30 min is recommended.

4. Allow the sample to cool to room temperature (approximately 10 min).

5. Add 25 μL of 360 mM IAA for a final concentration of 40 mM and incubate in the dark for 30 min.

6. Add 16.1 μL of 12% phosphoric acid for a final concentration of 1.2% and mix by pipetting.

7. Add 600 μL of freshly made S-trap binding buffer (see Recipes) and mix by pipetting. The solution will turn cloudy as the protein forms a colloid.

8. Label and place the S-trap spin column (cap must be loosely placed on top) on a 2 mL microcentrifuge tube to collect the waste.

9. Add 250 μL of sample from the collection tube into the S-trap spin column.

10. Centrifuge at 4,000× g for 30 s to pass samples through the S-trap spin column.

11. Continue to add samples (200 μL at one time with centrifugation after each step) until completely loaded onto the S-trap spin column.

Note: The maximum loading capacity of this S-Trap column is 100 μg in this study, as indicated in the reagents section. The choice of S-Trap column size is determined based on the protein amount after Top 14 protein depletion.

12. Wash the S-trap spin column with S-trap binding buffer (300 μL each time, three times in total).

13. Transfer the S-trap spin column to a new clean 2 mL microcentrifuge tube.

14. Add 115 μL of 50 mM ABC to the S-trap spin column.

15. Add 10 μL of trypsin solution, cap the S-trap spin column, put it on the rack, and then put it into the incubator at 37 °C for 18 h.

C2. Trypsin digestion (plate format)

1. Resuspend samples in 50 μL of 1× lysis buffer.

2. Using a multichannel pipette, add 5 μL of 110 mM DTT for a final concentration of 10 mM (the 110 mM DTT working solution can be prepared from DTT powder or by dilution from a 1 M DTT stock solution). Mix by pipetting.

3. Cover the deep-well plate with a sealing mat and heat at 95 °C for 15 min with shaking/mixing.

Note: Denaturation, reduction, alkylation, and acidification can be done in 1.5 mL low protein-binding tubes and then transferred to a S-Trap plate.

4. Allow to cool to room temperature (~10 min) (alternatively, centrifuge for 2 min).

5. Add 5 μL of 240 mM IAA (prepared fresh immediately before use; light-sensitive) for a final concentration of 20 mM and incubate in the dark for 30 min.

6. Add 5 μL of 32.5% phosphoric acid for a final concentration of 2.5%. Mix by pipetting.

7. Add 390 μL of freshly prepared S-Trap binding buffer and mix by pipetting. The solution will turn cloudy as proteins form a colloid.

8. Label the S-Trap plate and place it on a square-well collection plate to collect waste.

9. Add 250 μL of sample to the S-Trap plate at a time (repeat until the entire sample is transferred). Using the Tecan A200 Positive Pressure Manifold, apply increasing pressure (i.e., Flash) until all liquid has passed through. Alternatively, centrifuge the plate at 800× g for 2–5 min to pass the liquid through the membrane.

10. Wash 4 times with 300 μL of S-Trap binding buffer, applying positive pressure as in the previous step.

11. Place the S-Trap plate on top of a clean 2 mL deep-well collection plate.

12. Add 150 μL of 50 mM ABC containing sufficient trypsin (1:50 minimum enzyme to protein ratio; 1:100 may lead to incomplete digestion and large peptide fragments) using a single or multichannel pipette. Cover and place in a suitable ziplock bag with a damp Kimwipe to provide humidity and prevent evaporation of trypsin solution. Alternatively, cover the plate with a clear plastic lid and incubate, placing a pan of water in the incubator to prevent the plate from drying out. Incubate for 18 h at 37 °C.

D. Peptide elution from S-trap

D1. Elution (tube format)

1. Transfer the rack to room temperature. After removing the S-trap column cap on top of the S-trap spin column, add 40 μL of elution buffer 1 to the S-trap and then centrifuge at 4,000× g for 1 min.

2. Add 40 μL of elution buffer 2 to the S-trap and then centrifuge at 4,000× g for 1 min.

3. Add 40 μL of elution buffer 3 to the S-trap and then centrifuge at 4,000× g for 1 min). This elution assists in the recovery of hydrophobic peptides.

4. Pool eluted peptides, dry down, and resuspend in aqueous buffer (see Recipes) for proteomics data acquisition.

D2. Elution (plate format)

1. After removing the S-Trap plates, allow the plate to cool and reach room temperature before placing a collection plate spacer between the S-Trap plate and the collection plate.

2. Allow the A200 to dispense 50 μL of 50 mM ABC (or 50 mM TEAB digestion buffer). Then, apply positive pressure.

3. Add 50 μL of buffer A (see Recipes) and apply positive pressure.

4. Add 50 μL of buffer B (see Recipes), allow to stand for at least 5 min, and apply positive pressure.

5. Freeze the plate at -80 °C for at least 1 h before drying the plate using a SpeedVac concentrator at -4 °C.

6. Reconstitute when ready for LC/MS analysis using 5% acetonitrile and 0.1% formic acid.

E. Mass spectrometry proteomics

Data-independent acquisition (DIA) LC-MS proteomic analysis

Samples were separated using a Vanquish Neo UHPLC coupled to a Thermo Fisher Ascend Tribrid mass spectrometer. Samples were initially loaded onto an Acclaim PepMap C18 Trap column (7 cm total length, 2 cm bed length, 3 μm particle size, 100 A pore size) and IonOpticks Aurora Ultimate analytical column (25 cm long × 75 μm ID, 1.7 μm particle size) at a flow rate of 300 nL/min over a 60-min gradient. Solvent A consisted of LC-MS-grade water with 0.1% formic acid, and solvent B consisted of 80% acetonitrile and 0.1% formic acid. The gradient consisted of loading/starting conditions of 5% B, then to 30% B over 50 min, followed by 95% B over 5 min, finishing at 99% B for the remaining 5 min. A combined control was used for sample loading and washing. The trap column was washed using four zebra washes, and the analytical column was washed using three alternating aqueous and organic cycles before being re-equilibrated for the next sample injection. The mass spectrometry method consisted of a final spray voltage of 2,050 V and a charge state of 2. For the master scan, quadrupole isolation was selected, and Orbitrap resolution was set to 120K at a scan range of 350–1,650 m/z. A maximum injection time of 100 ms and a normalized AGC target of 50% were selected. RF lens was set to 30%, and the data type was set to profile mode. For the DIA scan, isolation utilized the quadrupole, with isolation widths of 20 m/z with a 4 m/z overlap, which led to 40 scan events. HCD fragmentation was used with a normalized CE of 28%, and the detector was set to Orbitrap at 30K resolution with a mass range of 360–1,160 m/z. Max injection time was set to 50 ms, and the AGC target was 1,000%.

Data analysis

Spectronaut version 19.4.241104.62635; analysis type: directDIA. Process and analyze raw data using the Spectronaut^®/DirectDIA (Biognosys) workflow with default settings. Detailed descriptions of the DirectDIA workflow have been provided in our previous publications [10,11].

By default, Spectronaut selects between local and global normalization algorithms based on the number of runs in an experiment: local normalization is applied when the number of runs does not exceed 500 (n< 500), while global normalization is used for larger experiments. In this study, the local normalization algorithm was employed. Rat UniProt FASTA database (UP000002494_10116_Rat_Reference_20240602.fasta) and Universal Contaminant Protein FASTA (created 06.01.22, used 06.03.25) were used. A false discovery rate (FDR) threshold of 1% was applied at the peptide, protein, and peptide–spectrum match (PSM) levels. Protein identifications were reported at the protein group level, and the total number of proteins was determined from protein groups passing the 1% protein-level FDR filter. This statistical FDR, together with mProphet scoring and IDPicker algorithms, minimizes potential mis-annotations. The extracted ion chromatogram (XIC) retention time window was set to dynamic, with tolerances automatically optimized by Spectronaut, and the correction factor was maintained at the system default.

Additional details regarding the DirectDIA workflow and underlying algorithms are available in the Spectronaut User Manual (https://biognosys.com/content/uploads/2023/03/Spectronaut-17_UserManual.pdf). The number of proteins identified in the 31 samples is listed in Table 1.

Table 1. Protein identifications in 24 rat cerebrospinal fluid samples

Validation of protocol

This protocol or parts of it has been used and validated in the following research articles:

1. Silasi et al. [12]. Protective effects of factor XI inhibition by abelacimab in a baboon model of live Staphylococcus aureus sepsis. Journal of Thrombosis and Haemostasis.

2. Jansen et al. [13]. Small molecule inhibition of ubiquitin C-terminal hydrolase L1 alters cell metabolism proteins and exerts anti- or pro-tumorigenic effects contingent upon chemosensitivity status in high grade serous ovarian cancer. Frontiers in Pharmacology.

3. Biedka et al. [14]. One-pot method for preparing DNA, RNA, and protein for multiomics analysis. Commun Biol.

4. Khadka [15]. Circulating microRNA Biomarker for Detecting Breast Cancer in High-Risk Benign Breast Tumors. Int J Mol Sci.

5. McCoy et al. [16]. Ovarian somatic tissue rejuvenates circulating apolipoproteins and promotes cognitive health in postreproductive female mice. GeroScience.

General notes and troubleshooting

General notes

1. Avoid multiple freeze/thaw cycles of biological samples to ensure protein integrity.

2. The peptides generated by this protocol are compatible with most liquid chromatography systems and mass spectrometry acquisition methods (e.g., DDA or DIA). DIA analysis was selected to evaluate the robustness of the S-Trap sample preparation method; however, the protocol is not limited to DIA and is equally applicable for sample preparation in DDA workflows.

3. This protocol is designed for biofluid sample preparation, including high-abundance protein depletion, digestion, and peptide cleanup. While Top 14 protein depletion improves the detection of low-abundance proteins, the protocol itself is compatible with both qualitative (identification) and quantitative proteomics analyses. The determination of protein quantification depends on the LC–MS/MS method employed.

4. Resulting peptides may also be enriched further using TiO₂ or bead-based enrichment protocols for PTM-specific analysis.

Troubleshooting

Supplementary information

The following supporting information can be downloaded here:

1. Supplementary Figure 1. Distribution of precursor identifications across 31 CSF samples.

2. Supplementary Table 1. Protein identifications and sample group information for 31 rat cerebrospinal fluid (CSF) samples

Acknowledgments

This research is based in part upon work conducted using the Proteomics Core Facility, which was supported in part by the National Institutes of Health Grant No. 1S10OD036295 (Ascend Tribrid, FAIMS, Vanquish Neo) and the Division of Biology and Medicine, Brown University. C.L. was supported by a Pilot Grant Award (2026) from the College of Pharmacy, University of Rhode Island. C.L. also received a training award (2025) in sample preparation and mass spectrometry data analysis from the IDeA National Resource for Quantitative Proteomics, supported by NIH grants P20GM103429, P20GM121293, and R24GM137786.

Competing interests

The authors have no competing interests to disclose.

Ethical considerations

This protocol was conducted in strict accordance with the University of Rhode Island’s Institutional Animal Care and Use Committee (IACUC).

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