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Method Article

Enriching Subcellular Proteins in Leptospira Using a Triton X-114-Based Fractionation Approach

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DOI:

10.3791/67298

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August 8th, 2025

 ,  ,  ,  ,  , 
1Department of Biochemistry, ICMR - Regional Medical Research Centre, 2Department of Chemistry & Molecular Biology, University of Gothenburg

In This Article

Summary

Though prokaryotes lack an organellar system, there are subcellular regions with localized proteins. Quantitative and qualitative analysis of the enzymes and proteins in the subcellular regions is important for developing drug and vaccine targets. Here, we illustrate the subcellular fractionation of Leptospiral proteins using Triton X-114 and its analysis.

Abstract

Prokaryotes lack precise subcellular organelles but exhibit distinct regions such as cytoplasm, inner membrane, periplasm, and outer membrane, where most biochemical and physiological functions are organized. Thus, understanding the functional characteristics of proteins necessitates elucidating their subcellular localization. However, extracting subcellular proteins from gram-negative bacteria poses challenges due to their complex phospholipid bilayer. Although Triton X-114 has shown promise in outer membrane protein (OMP) extraction, a concise protocol remains elusive. This protocol demonstrates a step-by-step workflow for extracting subcellular proteins using the spirochete Leptospira as a model. This technique, featuring subcellular fractionation and phase separation, yields distinct fractions for cytoplasmic, outer, and inner membrane proteins. The detergent Triton X-114 is well-suited for phase separation due to its optimal cloud point temperature (~22 °C) and low critical micelle concentration (CMC), enabling efficient extraction and purification of native proteins with minimal denaturation. Notably, the analysis reveals the efficiency in discriminating proteins from the inner and outer membranes distinct from the cytoplasm.

Introduction

Subcellular proteomic (SCP) analyses play a pivotal role in elucidating bacterial pathogenesis by analyzing the proteome within specific subcellular compartments of bacterial cells. This subcellular proteomics approach characterizes bacterial proteins within distinct compartments, reducing complexity by excluding highly abundant proteins and providing insights into their organization and proportional abundance1,2. For instance, employing subcellular fractionation and LC-MS/MS techniques, a study on Shewanella oneidensis unveiled a substantial portion of its proteome, furnishing crucial details regarding the compartment architecture of cell and protein abundance. This study serves as a foundational framework for subsequent investigations into subcellular organization within gram-negative bacteria3. In the realm of bacterial pathogenesis, delineating the bacterial proteome during in vivo infection is imperative for comprehending the mechanisms driving pathogenicity4. Investigating the proteome of intracellular Salmonella within epithelial cells across various infection stages has been instrumental in unveiling the adaptive strategies employed by bacterial pathogens within their intracellular milieu4,5. SCP strategies provide comprehensive insights into cellular dynamics by assessing protein abundance and translocation within various cellular compartments. This method facilitates the extensive identification of proteins from diverse bacterial compartments, enhancing the thorough analysis of bacterial proteomes6,7.

The subcellular distribution of proteins not only dictates their functional activity but also enhances their functional diversity8. For instance, enzymes exhibit varied regulatory mechanisms and activity levels depending on their subcellular localization. Transglutaminases (TGases), for instance, display distinct roles or regulatory mechanisms across different subcellular compartments in pollen tubes: cytoplasmic TGases incorporate primary amines into cytosolic proteins (e.g., actin and tubulin) in a Ca(2+)-dependent manner, interacting with the cytoskeleton; membrane TGases are associated with Golgi and plasma membranes, suggesting a role in exocytotic delivery; and cell wall-associated TGases regulate apical growth by colocalizing with cell wall markers. These functional distinctions are mediated by compartment-specific roles9. Furthermore, the repair of oxidized proteins in the bacterial envelope underscores the critical role of enzyme systems within specific cellular compartments exposed to reactive oxygen species10. Consider endothelial nitric oxide synthase (eNOS), whose subcellular localization is modulated by lipid modifications, influencing its interaction with diverse cellular membranes11. Enzyme compartmentalization maintains distinct environments conducive to interactions between enzymes and substrates, thus facilitating compartment-specific metabolic processes12.

Bacteria and other prokaryotes do not have clear subcellular organelles; there are distinct regions, including cytoplasm, inner membrane, periplasm, and outer membrane. Most of the biochemical and physiological functions in the organism are organized in various subcellar compartments13. So, subcellular localization of proteins is important to predict their functional characteristics. However, extracting subcellular proteins from gram-negative bacteria poses challenges due to their complex double-membraned phospholipid bilayer. Conventional extraction methods, employing detergents like sodium dodecyl sulfate (SDS) and additives such as urea, often compromise membrane protein integrity, impacting downstream analyses14,15. Moreover, the lipid composition plays a significant role in shaping the conformational alterations of membrane proteins. The success of solubilization depends on factors like the detergent-lipid-protein ratio, as the process involves detergent insertion into the lipid bilayer followed by protein solubilization16. The choice of detergent is critical for solubilizing membrane proteins effectively, considering factors like cloud point temperature and incubation time17. The lipid environment can influence the conformational changes of membrane proteins18,19.

Detergents are essential for extracting membrane proteins from biological membranes and ensuring their solubility in aqueous solutions, a prerequisite for subsequent protein purification. However, purifying membrane proteins is often challenging because they are removed from their native lipid membrane environment and placed in a detergent buffer, which only partially replicates the membrane's physical and chemical properties20. After extraction, membrane proteins are typically purified in subsequent processes using dialysis, protein precipitation and some chromatography methods as soluble proteins20,21. Phase separation offers a powerful alternative or complement to chromatography-based purification protocols and can be applied directly to solubilized proteins. Basically, phase separation allows integral membrane proteins to partition into detergent enrich phase and cytosolic proteins in the detergent-depleted aqueous phase20,22. Several detergents have been described as being used in the solubilization of membrane proteins in earlier studies20. These include the Triton family (Triton X-100 & Triton X-114), Tween family (Tween-20, & -80), anionic detergents, cationic detergents, polyoxyethylene glycol monoether detergents etc., but they have pros and cons in terms of proteins structure and function20. The physicochemical properties of these detergents greatly influence the phase partition and solubilization of membrane proteins20. Detergents are amphipathic molecules with a polar headgroup and a hydrophobic hydrocarbon chain. At low concentrations, they exist as monomers in water. Above the critical micelle concentration (CMC), they form micelles whose size depends on the detergent type, with the aggregation number indicating the number of molecules per micelle (also called as micelle molecular weight). Another important factor is "cloud point" which can be reached by changing the temperature of an aqueous micellar detergent solution that becomes turbid and eventually forms two distinct phases. This process is known as phase separation or cloud point extraction. A high CMC can yield more membrane proteins corresponding to high concentrations of detergent with weaker binding, which can be dialyzed against a buffer with a defined detergent concentration20. However, reaching a high CMC in most detergents requires a high cloud point temperature, which can affect the extracted protein structure and function and affect downstream processes. Even the high concentration of detergent in this phase might be harmful to protein and create problems in liquid handling for pipetting precise volume due to high viscosity. For example, triton X-100 has a high yield of membrane proteins in the detergent micelle above the cloud point temperature of 64-65 °C. To maintain the working temperature, 9%-23% (NH4)2SO4 or 16%-25% NaCl addition can be reduced to room temperature (RT), resulting in a high concentration of detergent, which likely affects protein stability. Similarly, Tween-based extraction uses a high cloud point temperature (76 °C for tween 20 and 93 °C for tween 80). Anionic detergent SDS is frequently used in protein applications but usually denatures proteins20. Researchers, aiming to efficiently extract membrane proteins, identified Triton X-114 as a classic detergent due to its optimal cloud point temperature of approximately 22 °C, ideal for membrane protein studies. First introduced by Bordier in 1981, Triton X-114 has since been widely used for membrane protein extraction and purification across diverse sources, including animal and plant tissues as well as microorganisms20,22. Triton X-114's low CMC prevents its removal by dialysis; however, its mild nature minimizes the denaturation of membrane proteins in the detergent-enriched phase. Additionally, Triton X-114 forms smaller micelles (n = 7-8) compared to Triton X-10020. James G. Pryde's study protocol employed Triton X-114 for membrane protein extraction from cultured mammalian cells. By using a cloud point temperature above 20 °C (incubation at 30 °C for 3 min), integral membrane proteins partitioned into the detergent-enriched phase, while peripheral and cytosolic proteins were recovered from the detergent-depleted aqueous phase22. Furthermore, David A. Haake et al. used Triton X-114 to extract outer membrane proteins (OMPs) from Leptospira interrogans, with and without CaCl2 as an additive23,24. Later studies confirmed that Triton X-114 effectively extracts OMPs from subcellular compartments without additives like glycerol or CaCl225,26. However, the inclusion of salts can increase detergent concentration, which may affect large-scale proteome studies using high-throughput mass spectrometry20. In our work on subcellular proteome analysis, we employed Triton X-114 coupled with tandem mass spectrometry, achieving significant enrichment across all subcellular compartments, including cytoplasmic, inner membrane, and outer membrane proteins25,26. The primary aim of the present protocol is to efficiently enrich subcellular compartments of a typical gram-negative bacterial cell while minimizing issues in downstream processes. Additionally, the protocol is designed to be cost-effective and time-efficient. Here, we elaborate on the lab-developed optimized protocol for subcellular protein isolation and discuss each bottleneck encountered during the extraction process.

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Protocol

NOTE: This protocol utilizes Leptospira interrogans for the subcellular protein extraction experiment. While this protocol can be adapted for use with other gram-negative bacteria, optimization may be necessary depending on the specific research objectives. Table of Materials and Table 1 provide the list of chemicals and equipment, along with the compositions of buffers and reagent solutions, respectively, while Table 2 outlines laboratory facilities.

1. Leptospira strain and culture conditions

  1. Obtain the L. interrogans serogroup Icterohaemorrhagiae serovar Copenhageni strain Fiocruz L1-130.
    NOTE: This strain was obtained from the Indian Council of Medical Research - Regional Medical Research Centre in Port Blair, India, a WHO collaborating center specializing in leptospirosis diagnosis, reference, research, and training.
  2. Grow Leptospira (inoculum containing ~1 × 106 cells/mL) in 50 mL of Ellinghausen McCullough Johnson Harris (EMJH) medium supplemented with 1% bovine serum albumin (BSA).
  3. Incubate at 30 °C until the culture reaches the mid-log growth phase (3.5 × 108 cells/mL). Test aliquots of the cultured medium on nutrient agar plates to ensure the medium remains uncontaminated.
    NOTE: Utilize dark field microscopy and a Petroff-Hausser counting chamber to count the cells. For details, refer to a previously published article27.

2. Cell harvesting

  1. After a 7 day incubation period, transfer three biological replicates of L. interrogans culture into centrifuge tubes in equal volumes (25 mL each).
    NOTE: Leptospira culture was handled inside a Laminar air flow hood in a BSL-2 laboratory facility during the transfer.
  2. Centrifuge all tubes at 2500 × g for 45 min at 4 °C. Carefully decant the supernatant into a beaker containing disinfectant and discard it. Use the cell pellet for the next step.
  3. Wash the cell pellets three times with phosphate-buffered saline (1 mL each time) supplemented with 5 mM MgCl2, centrifuging at 10,000 × g for 10 min at 4 °C after each wash.
    NOTE: This step will remove the unwanted BSA present in the EMJH medium from the surface of the cell pellet.

3. Subcellular protein extraction

  1. Triton X-114 extraction
    1. Treat the Leptospira cell pellet, obtained after harvesting, with an extraction buffer containing 10 mM Tris-Cl (pH 8), 1% Triton X-114, and 150 mM NaCl at a rate of 1 mL per pellet harvested (Table 1) (Optional -a protease inhibitor cocktail can add according to experimental purpose).
      NOTE: The procedure involves simply exposing bacteria to a detergent solution by adding the extraction buffer to extract membrane proteins.
    2. Gently mix the extraction buffer with the pellet using a micropipette below 25 °C until the solution becomes visually turbid.
    3. Incubate the mixture overnight at 4 °C.
    4. Mix the resulting extract again with a micropipette and centrifuge it at 15,000 × g for 30 min at 4 °C.
    5. Decant the supernatant into another vial and retain the cell pellet for subsequent extraction processes.
      NOTE: This supernatant contains a mixture of hydrophilic and hydrophobic proteins abundantly from the cytoplasm and outer membrane, and it is used for phase separation.
    6. Add 50 µL of buffer containing 10 mM Tris-Cl (pH 8), 8 M urea, a protease inhibitor cocktail, and 1% sodium dodecyl sulfate (SDS) to the cell pellet.
    7. Vortex the mixture continuously for 5 min after adding the extraction buffer, then incubate at 4 °C for another 5 min.
    8. Centrifuge the mixture again at 15,000 × g for 30 min at 4 °C and designate the resulting supernatant as the pellet fraction (P).
      NOTE: The pellet fraction was found to contain a high abundance of inner membrane proteins in Leptospira25,26.
  2. Phase partition
    1. Adjust the Triton X-114 concentration of the supernatant obtained from the extraction to 2%. For example, if the supernatant volume is 900 µL, add 9 µL of Triton X-114 detergent to achieve the desired concentration.
    2. Incubate the mixture at 37 °C for 1 h, then centrifuge at 2000 × g for 5 min at 30 °C. This low-speed centrifugation can accelerate the separation of phases.
    3. Allow the mixture to separate into two distinct phases: aqueous and detergent. Ensure proper centrifugation at low speed to achieve visible separation. If necessary, allow it to sit for 1 min before isolation. If separation does not occur, repeat the step 3.2.2 as needed.
      NOTE: Phase separation can be observed visually, by turbidity measurements using a spectrophotometer, or through static light scattering. To increase the accuracy of the cloud point assay, add a hydrophobic dye (5-Hexadecanoyl-aminoeosin) with a concentration of 500 nM to the solution and analyze it with ultraviolet-visible spectroscopy (UV-VIS).
    4. Isolate the upper aqueous phase by piercing the interface with a sterile syringe and transferring it to a separate vial. Isolate the bottom detergent phase using a micropipette and store it in a separate vial.
      NOTE: Handle the vial containing both phases carefully during the separation process. Before piercing the vial with a syringe, open its lid to prevent needle breakage or difficulty in extracting the upper aqueous phase. Using a syringe ensures more accurate volume measurement of each phase after separation and minimizes protein cross-contamination between the phases.
    5. Label the phases as aqueous (A) and detergent (D) and store them at -20 °C for future use.

4. Protein estimation

  1. Assess the protein concentrations of each Triton X-114 fraction (aqueous, detergent, and pellet) using the bicinchoninic assay28 or any other suitable methods.

5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) gel electrophoresis

NOTE: Proteins from each fraction (aqueous, detergent, pellet) must be treated with beta-mercaptoethanol to reduce them, then separated using 10% SDS-PAGE (three biological replicates). The recipe for the 10% SDS-PAGE can be found in Table 1. For protein sample targeting, any enzyme activity analyses need to be done in a native form that can be resolved in native PAGE.

  1. Resuspend the protein sample in ice-cold acetone using four times the volume of the sample. Incubate the mixture at -20 °C for a minimum of 2 h, then centrifuge at 14,000 × g for 10 min. Additionally, resuspend the pellet fraction in 80% ice-cold acetone, then incubate for 5 min at 4 °C. Centrifuge at 14,000 × g for 10 min to remove excessive urea salt crystals.
  2. After centrifugation, carefully remove the supernatant and place the protein pellet in a dry bath until all residual acetone evaporates from the vials.
  3. Incubate the protein samples in 20 µL sample buffer containing beta-mercaptoethanol, then heat them at 95 °C for 10 min in a water bath or dry bath.
    NOTE: Here, the protein samples represent the aqueous (A) fraction containing cytoplasmic proteins, the detergent (D) fraction containing outer membrane proteins, and the pellet (P) fraction containing inner membrane proteins.
  4. Load the samples containing A, D, and P into each well of the 10% SDS-PAGE gel alongside a prestained protein marker (M) and total protein (T).
    NOTE: The total protein can be prepared by pulling all three fractions together into a single vial by using 20 µL of sample buffer.
  5. Use 1x electrode buffer in the electrophoresis apparatus (Table 1). Set the gel running parameters to 6 mA for the stacking gel and 12 mA for the resolving gel. Run the electrophoresis at RT.
  6. Stain the gel with Coomassie Brilliant Blue G or other protein stains available in the lab.

6. Immunoblotting

  1. After gel separation, incubate the unstained gel in transfer buffer (25 mM Tris base, 190 mM glycine, 20% methanol) for 5 min.
  2. Incubate appropriately sized blotting papers in immunoblot transfer buffer for 5 min, then assemble them as the bottom layer of the sandwich.
  3. Utilize polyvinylidene fluoride (PVDF) membranes or nitrocellulose membranes to transfer proteins from the unstained gels. Begin by immersing the PVDF membrane in absolute methanol for 30 s, followed by a 5-min soak in the immunoblot transfer buffer. Subsequently, integrate it into the blotting sandwich.
  4. Now, place the unstained gel that has been removed from the transfer buffer and use it in the transfer blotting sandwich.
  5. Finally, cover the sandwich by placing it on other blotting paper on top of it, release any trapped air bubbles with a blot roller, and run the transfer using a semi-dry transfer method.
    NOTE: Other approaches, including dry and wet transfer, can be used depending on laboratory facilities.
  6. Submerge the membrane in a blocking buffer (8% non-fat milk in TBS-0.1% Tween 20 [TBS-T]) for 2 h, followed by overnight incubation at 4 °C with monospecific antisera* targeting Leptospira lipoprotein LipL41 (1:5000), or flagellar filament 35 kDa core protein (FlaB) (1:4000) (*custom antibody). Rinse the membrane three times with TBS-T, allowing each rinse to proceed for 5 min.
    NOTE: Use any antibody of interest. The dilution factor of antibodies may vary.
  7. Incubate the membrane with a mouse anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP) IgG (1:20000 dilution) for 2 h at RT.
  8. Expose the membrane to the electrochemiluminescence (ECL) reagent to visualize protein bands.
  9. Place the membrane under blue-sensitive X-ray film within a dark room.
    NOTE: Any other developing methods also can be utilized.

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Results

In this experiment, we employed the spirochete Leptospira as a model organism to extract subcellular compartments. We employed a strategic methodology anchored in cloud point and critical mycelial concentration (CMC), harnessing the effectiveness of Triton X-114, a non-ionic detergent (Figure 1). Before proceeding further, protein estimation was conducted to assess the concentration in each fraction for a single biological replicate. We observed a concentration ratio of 3:1:1.3 in A...

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Discussion

Subcellular fractionation techniques play a pivotal role in the isolation of discrete cellular constituents for subsequent analysis. Triton X-114, a non-ionic detergent, is commonly used in these techniques because of its ability to partition samples into distinct phases, allowing for the separation of various cellular components32. Triton X-114 has been particularly useful in extracting membrane proteins (in native form) from a variety of sources, including urine extracellular vesicles, Golgi mem...

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Disclosures

The authors have declared that they have no conflicting financial interests.

Acknowledgements

The authors express their sincere gratitude to the Indian Council of Medical Research, New Delhi, India, for the financial support provided through Grant No. Leptos/22/2013-ECD-I-2012-2400 and the Department of Science and Technology, Science and Engineering Research Board, New Delhi, India, for their funding support via Grant No. SR/SO/HS/0108/2012 to M.G.M. Additionally, the authors extend their appreciation to the Indian Council of Medical Research, New Delhi, India, for awarding a Senior Research Fellowship through Grant No. ICMR-SRF 2020-0756/PROTEOMICS- BMS, Fellowship/131/2022-ECD-II-2021-8230 to H.P and AS respectively and Department of Science and Technology, Science and Engineering Research Board, New Delhi, India, Grant No. PDF/2017/000343 to S.A.D.C.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Acetic acid  Sigma 695092
Acetone  Sigma 32201
Acrylamide  Sigma A-3553
Ammonium persulfate  Sigma A-3678
Balance SHIMADUZ Z741079
Bis Acrylamide  Sigma M7279
Bovine serum albumin fraction V (BSA)  Sigma A-8022
Bromophenol Blue  Sigma B-8026
Centrifuge Tube (50 mL)Tarsons500020
Conical Flask with Cap (150 mL)Tarsons431030
Cooling centrifuge Eppendorf 5355316200
Coomassie brilliant blue  Sigma1.12553
Dark field microscope ZEISS GmbH37081
Delicate Task WipesKimtech Science34155
Dry bath Eppendorf 535531620
Ellinghausen–McCullough–Johnson–Harris (EMJH) medium, Difco Difco 279410
Gel trays: Gel trays must be at least 2cm wider in dimensions and have smooth and flat surface with lid.  Hoefer Not applicable
Glass Beake (150 mL)Tarsons421030
Glycine G8790  Sigma G-8898
Immobilon Blotting Filter PaperMilliporeIBFP0813C
Immunoblot semi-dry setup ATTA AE-6675
Incubator - For incubating the gel trays for 18 hours at 37°C. EYELA SLI-700
Laminar air flow Not applicable Not applicable
Luminol  Sigma 09253
Magnesium chloride M8266  Sigma M8266
Magnetic stirrer TARSONS 6040
Methanol  Sigma 17995-7
Micropipette Tips (10 µL–10 mL)AxygenT-10, T-200-C,T-1000-C, T-10ML-C
Micropipettes Eppendorf EP2231300010
Microtubes (1.5 mL Safe-Lock)EppendorfEP022363212
Mini spin Genaxy GEN-MINI-6K
Multimode readerBMG LABTECH S/N 413-3877
Nonfat-Dried Milk, BovineSigma-AldrichM7409
P-Coumaric acid  Sigma C9008
Phosphate buffered saline  SigmaP4417 
Refrigerator LG GC-B217BLJ2
Rocker - Rocking or rotary shaker with low speed for staining the gel.  TARSONS 4080
SDS-PAGE SETUP- Electrophoresis Apparatus Hoefer, Inc. SE300-10A-1.0 Hoefer model ‘miniVE’ which can cast 10 cm x 8 cm sized gel of thickness 1 mm. 
Sodium chloride   Sigma 5886
Sodium dodecyl sulfate (SDS)  Sigma L-4396
TEMED  Sigma T-9281
Triton X-114  Sigma648468
Trizma Base (TRIS) T6066  Sigma T6066
Vortex TARSONS 3020

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Subcellular FractionationTriton X 114 ExtractionLeptospira ProteinsOuter Membrane ProteinsInner Membrane ProteinsPhase SeparationProtein LocalizationSDS PAGEImmunoblottingMass Spectrometry
Enriching Subcellular Proteins in Leptospira Using a Triton X-114-Based Fractionation Approach
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