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Isolation of Exosomes for the Purpose of Protein Cargo Analysis with the Use of Mass Spectrometry

  • Monika Pietrowska
  • Sonja Funk
  • Marta Gawin
  • Łukasz Marczak
  • Agata Abramowicz
  • Piotr Widłak
  • Theresa Whiteside
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1654)

Abstract

Exosomes are intercellular messengers with a high potential for diagnostic and therapeutic utility. It is believed that exosomes present in body fluids are responsible for providing signals which inhibit immune cells, interfere with antitumor immunity, and thus influence the response to treatment and its effect. One of the most interesting issues in exosome studies is proper addressing of their cargo composed of nucleic acids and proteins. Effective and selective isolation of extracellular vesicles and identification of proteins present in exosomes has turned out to be a challenging aspect of their exploration. Here we propose a novel approach that is based on isolation of exosomes by mini-size-exclusion chromatography which allows efficient, rapid, and reliable isolation of morphologically intact and functionally active exosomes without the need of ultracentrifugation. The purpose of this chapter is to describe a simple and high-throughput method to isolate, purify, and identify exosomal proteins using a mass spectrometry approach. The proposed protocol compiles the expertise of two research groups specialized in exosome research and in mass spectrometry-based proteomics. The protocol combines differential centrifugation followed by ultrafiltration, centrifugation-based filtration, and gel filtration on Sepharose 2B in order to obtain exosomal fractions characterized by only low contamination with albumin.

Key words

Albumin removal Cell culture Exosomes Filter-aided sample preparation Mass spectrometry Peptide assay Proteomics Size exclusion chromatography Ultrafiltration 

1 Introduction

Exosomes are double-layer membrane vesicles having a diameter of several tens of nanometers which are formed in late endosomes otherwise known as multivesicular bodies (MVBs) [1]. There are several lines of evidence that exosomes, released by both tumor and non-tumor cells, may be key players involved in intercellular communication. Moreover, their ability for presentation of antigens and modulation of the immune response as well as possible role in development of some neurodegenerative diseases make them a very attractive subject of molecular studies [2]. However, there are still many missing pieces of the puzzle that should be discovered for full understanding of the biological activity and function of exosomes. One of the essential research areas in the exosomes field is characterization of their protein cargo. Previous proteomic studies led to the identification of numerous proteins, either constitutive or occasionally occurring molecules that may be crucial for specific functions of these vesicles. Analysis of exosomes from a wide variety of cells and body fluids has allowed for identification of several functional classes of proteins: membrane adhesion factors, membrane transport/trafficking factors, cytoskeletal components, lysosomal markers, antigen presenting factors, cancer-specific antigens, death receptors, cytokines and cognate receptors, iron transport factors, metabolic enzymes, heat shock proteins, and drug transporters. The presence of specific proteins in tumor-derived exosomes suggests the existence of a protein sorting mechanism during their formation [3]. The presence of specific proteins can reflect the origin of exosomes and their functional role. Proteins present in tumor cell-derived exosomes can be a useful source of cancer biomarkers. It has been shown that exosomes released in vitro from breast carcinoma cells contain HER2, while carcinoembryonic antigen was found in exosomes secreted from colon carcinoma cells. Moreover, MelanA/Mart-1 and gp100 proteins that are expressed in melanoma cells were also found in released exosomes [4]. It was observed that amount and content of exosomes isolated from serum [5], ascites fluids [6], pleural effusions [4], and urine [7] of cancer patients positively correlated with tumor progression. There is no doubt that in-depth characterization of the proteomes of vesicles derived from different types of cancer cells could bring a relevant and timeless knowledge in the field of molecular oncology. However, there are two major challenges in studies focused on exosomal proteomes quantity and purity of the analyte. There are several popular methods of exosome isolation like ultracentrifugation, ultrafiltration, or immuno-capture. All of them have some specific features making them more or less suitable for mass spectrometry applications (reviewed in details in [8]).

Effective preparation and quantification of proteins/peptides for mass spectrometry analysis is an important aspect in processing of biological material. It is crucial for peptide identification yield and for the success of the whole experiment. Unfortunately, a substantial loss of analyte is unavoidable in all processes of sample preparation/purification, which is especially undesirable in the case of low-abundant analytes. At the same time, exact quantification of proteins/peptides intended for mass spectrometry analysis is crucial for the credibility of results, especially when using a label-free strategy. As a consequence, it often means working with trace amounts of biological material, insufficient for the flagship methods of protein quantification like Bradford or BCA assays. Mass spectrometry is an analytical technique especially suited for analyses of exosomes due to a small amount of available protein material. However, the quality of isolated exosomes is a crucial condition of a successful analysis.

Here we propose a complete and validated protocol for preparation of high quality samples of exosomal proteins for high resolution mass spectrometry analysis. It is a high-throughput method suitable for exosome isolation without the loss of their biological activity (Fig. 1). The pipeline includes well-known steps modified and adapted to low-scale vesicle studies, sensitive protein/peptide quantification by tryptophan fluorescence measurement, protein digestion according to a modified FASP method originally introduced by Wiśniewski et al., and finally qualitative and quantitative analysis by LC-MS/MS resulting in high coverage of a sample proteome.
Fig. 1

Preparation of albumin-depleted exosomal fractions

2 Materials

2.1 Laboratory Equipment

  1. 1.

    Centrifuge equipped with a fixed angle rotor for 1.5/2 ml centrifuge tubes and with adaptors for 50 ml centrifuge tubes, and a swing bucket rotor with adaptors for 250 ml bottles (for Vivacell 100 concentrators).

     
  2. 2.

    Temperature controlled shaker.

     
  3. 3.

    Vortex mixer.

     
  4. 4.

    Vacuum concentrator.

     
  5. 5.

    Laboratory incubator (working at 37 °C, 5% CO2).

     
  6. 6.

    Microplate reader enabling fluorescence excitation in the range of UV light and fluorescence detection in visible light range.

     
  7. 7.
    LC-MS/MS system:
    1. (a)

      MALDI-ToF/ToF MS ultrafleXtreme™ coupled with EASY nLC nano-liquid chromatograph and PROTEINEER fc II fraction collector (all from Bruker Daltonik).

       
    2. (b)

      Hybrid mass spectrometer Q Exactive Quadrupole-Orbitrap coupled with Dionex Ultimate 3000 RSLC nanoLC (both from Thermo Fisher Scientific).

       
     

2.2 Isolation of Exosomes for Mass Spectrometry Application

2.2.1 Solutions/Reagents

  1. 1.

    Cell culture medium (e.g., Dulbecco’s Modified Eagle’s Medium—high glucose, further referred to as DMEM).

     
  2. 2.

    Fetal bovine serum free from animal exosomes (FBS EXO-) (e.g., Gibco™ Exosome depleted FBS, Thermo Fisher Scientific).

     
  3. 3.

    Phosphate buffered saline (PBS).

     
  4. 4.

    Penicillin-Streptomycin (10,000 U/ml).

     
  5. 5.

    Sepharose® 2B 60–200 μm bead diameter (Sigma-Aldrich).

     
  6. 6.

    Lysis buffer (exoLB): 6% sodium dodecyl sulfate (SDS), 200 mM dithiothreitol (DTT), 200 mM Tris–HCl pH 7.6.

     

2.2.2 Consumables

  1. 1.

    150 cm2 cell culture flasks, sterile.

     
  2. 2.

    50 ml centrifuge tubes.

     
  3. 3.

    Disposable plastic Pasteur pipettes.

     
  4. 4.

    0.22 μm syringe filters with hydrophilic membrane.

     
  5. 5.

    Vivacell® 100 concentrator units, 100,000 MWCO, PES membrane (Sartorius).

     
  6. 6.

    Econo-Pac® chromatography columns (Bio-Rad) (see Note 1 ).

     

2.3 Protein/Peptide Assay by Tryptophan Fluorescence Method

  1. 1.

    l-Tryptophan, stock solution: 1 mg/ml in water; working solutions in water: 0.1 mgTrp/ml and 0.01 mgTrp/ml (see Note 2 ).

     
  2. 2.

    96-Well or 384-well non-treated black flat-bottom polystyrene microplate.

     

2.4 Protein Digestion and Fractionation

For additional details concerning preparation of solutions, SAX-tip columns, and desalting C18-tip columns for mod-FASP, please refer to [9, 10] and instructions given by the Authors at the webpage of the Max Planck Institute of Biochemistry [11, 12].

2.4.1 Solutions

  1. 1.

    8 M urea in 0.1 M Tris–HCl pH 8.5.

     
  2. 2.

    50 mM iodoacetamide (IAA) in 8 M urea/0.1 M Tris–HCl pH 8.5, or 100 mM IAA in ultrapure water for in-solution digestion.

     
  3. 3.

    Trypsin (Promega): stock solution 0.5 μg/μl in 50 mM acetic acid for mod-FASP protocol, or 0.1 μg/μl in 50 mM acetic acid for in-solution digestion (see Note 3 ).

     
  4. 4.

    50 mM Tris–HCl pH 8.5.

     
  5. 5.

    For in-solution digestion: 100 mM DTT in ultrapure water, and 50 mM NH4HCO3 in ultrapure water.

     
  6. 6.

    Methanol (at least HPLC gradient grade).

     
  7. 7.

    1 M NaOH.

     
  8. 8.

    Britton-Robinson Universal Buffer (BRUB) pH 5 and pH 2 (both diluted five times with water before use).

     
  9. 9.

    Solutions of trifluoroacetic acid (TFA) in water: 0.1% (v/v), 1% (v/v), 10% (v/v).

     
  10. 10.

    60% acetonitrile, 0.1% TFA (v/v) in water.

     

2.4.2 Consumables and Other Materials

  1. 1.

    Microcon-30 kDa Centrifugal Filter Unit with Ultracel-30 membrane (Merck).

     
  2. 2.

    Clear polypropylene 200 μl pipette tips, 0.5 and 2 ml reaction tubes (see Note 4 ).

     
  3. 3.

    Scalpel.

     
  4. 4.

    Empore™ SPE Disks Anion-SR, diam. 47 mm (SUPELCO).

     
  5. 5.

    Empore™ SPE Disks C18, diam. 47 mm (SUPELCO).

     
  6. 6.

    Blunt HPLC needle (Hamilton NDL KFga16/51mm/pst3) and a plastic or metal wire.

     
  7. 7.

    Humid chamber (see Note 5 ).

     

2.5 Mass Spectrometry

  1. 1.

    Highest quality plastic consumables and glass vessels, including test tubes, vials, pipette tips, and bottles for solution storage (see Note 4 ).

     
  2. 2.

    LC-MS grade solvents: water, acetonitrile (ACN).

     
  3. 3.
    For LC-MALDI MS:
    1. (a)

      NS-MP-10 Biosphere C18 pre-column (100 μm × 2 cm, 5 μm granulation, 100 Å) from Nanoseparations (Nieuwkoop, the Netherlands).

       
    2. (b)

      Acclaim PepMap100 C18 analytical column (75 μm × 15 cm, 3 μm granulation, 100 Å, Thermo Scientific).

       
    3. (c)

      MTP AnchorChip 1536 T F 800 μm target plate (Bruker).

       
    4. (d)

      α-Cyano-4-hydroxycinnamic acid (HCCA) matrix for MALDI-TOF MS.

       
    5. (e)

      Peptide Calibration Standard II for Mass Spectrometry (Bruker).

       
    6. (f)

      0.05% TFA/H2O.

       
    7. (g)

      90% ACN, 0.05% TFA.

       
     
  4. 4.
    For quadrupole-Orbitrap LC-MS/MS:
    1. (a)

      Acclaim PepMap100 C18, 5 μm, 100 Å, 300 μm i.d. × 5 mm (Thermo Scientific).

       
    2. (b)

      Acclaim PepMap RSLC C18, 2 μm, 100 Å, 75 μm i.d. × 25 cm, nanoViper (Thermo Scientific).

       
    3. (c)

      Pierce™ LTQ ESI Positive Ion Calibration Solution (Thermo Fisher Scientific).

       
    4. (d)

      0.1% formic acid/H2O.

       
    5. (e)

      90% ACN, 0.1% formic acid.

       
    6. (f)

      98% H2O, 0.1% TFA.

       
     

3 Methods

3.1 Isolation of Exosomes for Mass Spectrometry Application

  1. 1.

    Culture HNSCC cell lines in 25 ml DMEM (supplemented with 10% FBS EXO- and 1% Penicillin/Streptomycin) from 30–40% to 70–80% confluency in 150 cm2 cell culture flasks and collect supernatants after 72 h (see Note 6 ).

     
  2. 2.

    Centrifuge 10 min at 2000 × g, room temperature (for dead cells removal).

     
  3. 3.

    Collect the supernatant carefully (you can use a Pasteur pipette) and transfer it to a fresh centrifuge tube (see Note 7 ).

     
  4. 4.

    Centrifuge 30 min at 10,000 × g, 4 °C (for cell debris removal).

     
  5. 5.

    Collect the supernatant carefully (you can use a Pasteur pipette, you should leave some liquid on the bottom).

     
  6. 6.

    Filter the collected supernatant using a 0.22 μm syringe filter (for apoptotic bodies and microvesicles removal).

     
  7. 7.

    Prepare a Vivacell 100 concentrator (see Note 8 ).

     
  8. 8.

    Add 50 ml of the collected filtrate into a Vivacell 100 concentrator unit and centrifuge at 700 × g, 35 min, 4 °C, until the concentrate reaches exactly 1 ml.

     
  9. 9.

    Collect the concentrate containing exosomes from the upper chamber (1 ml).

     
  10. 10.

    Immediately load the exosome suspension onto a chromatography column filled with Sepharose 2B (see Note 1 ).

     
  11. 11.

    Elute exosomes and proteins with PBS. Collect 1 ml per one fraction.

     
  12. 12.

    Perform identification of exosomes in the enriched fractions (usually fractions 3–4) using Western Blot analysis (see Note 9 ).

     
  13. 13.

    Lyse exosomes with the use of exoLB in the ratio of 2:1 (v/v) and incubate the solution at 95 °C for 5 min. Perform protein assay with the use of tryptophan fluorescence method.

     

3.2 Protein/Peptide Assay by Tryptophan Fluorescence Method

A versatile method of protein/peptide assay is needed at different stages of the protocol: from extraction of exosomal proteins to mass spectrometry analysis. For this purpose we adapted the tryptophan fluorescence method as proposed by Wiśniewski and Gaugaz [13]. A set of standards of l-tryptophan is prepared based on the assumption that the content of tryptophan in animal tissues is 1.17% and taking into account the buffer proteins/peptides are dissolved in, i.e., the sample matrix (see Notes 10 and 11 ).
  1. 1.

    Prepare a set of standards of l-tryptophan.

     
  2. 2.
    Load both standard solutions and protein/peptide samples into the wells of a selected micro-well plate (for sample volume below 50 μl you can use a 384-well plate) and measure tryptophan fluorescence in the conditions listed below:
    • Excitation: 295 nm, 5 nm bandwidth.

    • Emission: 350 nm, 20 nm bandwidth.

    • Temperature: 25 °C.

    • Top optic.

    • Individual measurements: 30 reads, 50 μs integration time.

    • Before each measurement: orbital-type shaking for 5 s followed by 2 s resting time.

    • Z-position to be set manually (e.g., 18,000 μm).

    • Detector gain to be set manually (e.g., for the most concentrated standard).

     
  3. 3.
    Perform at least three measurement series. Construct a calibration graph plotting fluorescence vs protein/peptide concentration or total protein/peptide content. The calibration dependence should be linear. Determine protein/peptide concentration in your sample from the calibration equation, always taking into account the limit of quantification (LOQ) of the method calculated as:
    $$ \mathrm{LOQ}=\frac{10\times {\mathrm{SD}}_{\mathrm{bl}}}{S} $$
    where SDbl is the standard deviation for a blank and S is the slope of a calibration plot.
     
  4. 4.

    Transfer each sample from a micro-well to a test tube for further processing.

     

3.3 Protein Digestion and Fractionation

Depending on the result of a protein assay and the kind of a buffer/solution the proteins are dissolved in, three procedures can be performed (Fig. 2). In our protocol protein extracts are subjected to a modified version (mod-FASP) of the multiple-enzyme digestion filter-aided sample preparation procedure (MED-FASP) proposed by Wiśniewski et al. [9, 10]. The latter procedure will not be described here and interested readers should refer to the original papers and educational materials provided by these authors [11, 12].
Fig. 2

Possible workflows of sample preparation for mass spectrometry analysis of exosomal proteins

3.3.1 Modified Filter-Aided Sample Preparation (Mod-FASP)

  1. 1.

    Prepare tip columns: you will need one SAX-tip column and two C18-tip columns per sample (see Note 12 ).

     
  2. 2.

    Load up to 50 μl of exosomal protein extract and 200 μl of 8 M urea in 0.1 M Tris–HCl pH 8.5 into a Microcon spin ultrafiltration unit. Centrifuge for 15 min at 14,000 × g (room temperature, RT).

     
  3. 3.

    Add 200 μl of 8 M urea in 0.1 M Tris–HCl pH 8.5 (100 μl), centrifuge for 15 min at 14,000 × g (RT).

     
  4. 4.

    Add 50 μl of iodoacetamide solution (50 mM), mix in a shaker for 1 min at 600 rpm (RT). Incubate in darkness for 20 min. Centrifuge for 15 min at 14,000 × g (RT).

     
  5. 5.

    Add 100 μl of 8 M urea in 0.1 M Tris–HCl pH 8.5, centrifuge for 15 min at 14,000 × g (RT). Repeat this step twice.

     
  6. 6.

    Add 100 μl of 50 mM Tris–HCl pH 8.5, centrifuge for 15 min at 14,000 × g (RT). Repeat this step twice.

     
  7. 7.

    Replace the collection tube with a new one. Add 40 μl of 50 mM Tris–HCl pH 8.5 with trypsin (enzyme-to-protein ratio of 1:100, w/w), mix in a shaker for 1 min at 600 rpm. Incubate in a humid chamber at 37 °C for 18 h (see Note 5 ).

     
  8. 8.

    Centrifuge the filter units for 15 min at 14,000 × g (RT).

     
  9. 9.

    Add 160 μl water and centrifuge again (15 min, 14,000 × g, RT).

     
  10. 10.

    Dilute thus obtained tryptic peptides with 200 μl of diluted BRUB pH 5.

     
  11. 11.

    Precondition SAX-tip columns via consecutive washes with: 100 μl methanol, 100 μl 1 M NaOH, 100 μl of diluted BRUB pH 5, and again 100 μl of diluted BRUB pH 5; follow addition of each solution by centrifugation: 4000 × g, 5 min (RT).

     
  12. 12.

    Precondition C18-tip columns via consecutive washes with: 50 μl methanol, 50 μl 60% ACN/0.1% TFA, and 50 μl 0.1% TFA/H2O; follow addition of each solution by centrifugation: 4000 × g, 5 min (RT).

     
  13. 13.

    Insert a SAX-tip column in a C18-tip column; load tryptic peptides: 2 × 200 μl, each loading followed by centrifugation: 5000 × g, 3 min (RT).

     
  14. 14.

    Add 100 μl of diluted BRUB pH 5, centrifuge: 5000 × g, 3 min.

     
  15. 15.

    Transfer the SAX-tip column to the next C18-tip column; add 100 μl of diluted BRUB pH 2, centrifuge: 5000 × g, 3 min (RT).

     
  16. 16.

    Discard SAX-tip column; wash C18-tip columns with 50 μl of 0.1% TFA/H2O (centrifugation at 5000 × g, 3 min, RT).

     
  17. 17.

    Elute peptides from C18-tip columns with 50 μl of 60% ACN/0.1% TFA (centrifugation at 5000 × g, 3 min, RT).

     
  18. 18.

    Remove the elution buffer from peptide fractions in a vacuum concentrator (see Note 11 ). Reconstitute with water (50 μl or less if you expect low peptide content).

     
  19. 19.

    Determine peptide content in the fractions using the tryptophan fluorescence method (refer to Subheading 3.2).

     
  20. 20.

    Before LC-MS/MS analysis acidify peptide fractions with 1% TFA/H2O (v/v) to reach the final TFA concentration of ca. 0.1% (v/v).

     
  21. 21.

    Dilute pH 5 peptide fraction with 0.1% TFA in order to achieve equal peptide concentration in both fractions. Equal fraction volumes need to be loaded onto an LC-column for each sample to maintain constant measurement conditions.

     

3.3.2 In-Solution Digestion

This protocol is adapted to a total sample volume of up to 10 μl. The final reaction mixture volume is 30 μl.
  1. 1.

    Mix 15 μl of 50 mM NH4HCO3 and 1.5 μl of 100 mM DTT in a 0.5 ml centrifuge tube.

     
  2. 2.

    Add the protein solution and adjust the final volume of the mixture to 27 μl with ultrapure water.

     
  3. 3.

    Incubate the mixture at 95 °C for 5 min, cool down to room temperature.

     
  4. 4.

    Add 3 μl of 100 mM IAA to the tube and incubate in the dark at room temperature for 20 min.

     
  5. 5.

    Add a proper volume of 0.1 μg/μl trypsin to the mixture to reach enzyme:protein ratio of 1:100 w/w (e.g., 1 μl for 10 μg of protein), incubate at 37 °C for 18 h.

     
  6. 6.

    Terminate the reaction by adding 1.5 μl 10% TFA.

     

3.4 Mass Spectrometry

Instrument settings given below should be considered only as a starting point for your MS method development. The optimal measurement conditions for your system may vary and should be adjusted according to your samples (see Notes 13 17 ). Depending on the peptide concentration in protein digests one should employ a sufficiently sensitive mass spectrometer (e.g., quadrupole-Orbitrap for the total peptide content below 5 μg). Nevertheless, if contaminants (e.g., high abundant proteins) are expected in a sample, application of a highly sensitive system may have the opposite effect to the intended one. Strictly speaking, increase in sensitivity of an MS system may not result in increase in the number of identified proteins.

3.4.1 LC-MALDI MS/MS

  1. 1.
    Nano-LC conditions:
    • Buffer A: 0.05% TFA/H2O.

    • Buffer B: 90% ACN, 0.05% TFA.

    • Pre-column: C18, 100 μm × 2 cm, 5 μm granulation, 100 Å.

    • Analytical column: C18, 75 μm × 15 cm, 3 μm granulation, 100 Å.

    • Acetonitrile gradient: from 2 to 45%, in 0.05% TFA.

    • Flow rate: 300 nl/min (113 min).

     
  2. 2.
    Fraction collection on a MALDI target plate:
    • MTP AnchorChip 1536 target plate.

    • α-Cyano-4-hydroxycinnamic acid (HCCA) solution (see Note 18 ).

    • Eluate from the analytical column is mixed with HCCA solution and spotted over 680 fractions on an MTP AnchorChip 1536 target plate.

     
  3. 3.
    MS and MS/MS conditions:
    1. (a)

      Collect MS spectra in positive reflector mode within tryptic peptide range (800–4000 m/z), 3000 shots from each LC fraction, random walk activated.

       
    2. (b)

      Fragment ions with S/N higher than 10, sum up 5000 shots for a fragment (MS/MS) spectrum.

       
     
  4. 4.
    Database search:
    1. (a)

      Use a selected program for database search and protein identification—we recommend Mascot Server 2.5.1 (Matrix Science, London, UK) and ProteinScape 3.1 (Bruker).

       
    2. (b)

      Set up proper search conditions: e.g., Swiss-Prot human database with a precision tolerance of 50 ppm for peptide masses and 0.5 Da for fragment ion masses; one missed cleavage; select Carbamidomethyl (C) and Oxidation (M) as fixed and variable modifications, respectively.

       
    3. (c)

      When using ProteinScape software you can perform protein list compilation by ProteinExtractor: ions score cutoff, peptide rank cutoff, and minimum peptide length set at: 15.0, 10, and 5, respectively; identity score calculated by the search engine.

       
     

3.4.2 Quadrupole-Orbitrap LC-MS/MS

  1. 1.
    Nano-LC conditions:
    • Buffer A: 0.1% formic acid/H2O.

    • Buffer B: 90% ACN, 0.1% formic acid.

    • Loading buffer for trapping: 98% H2O, 0.1% TFA.

    • Trapping column: C18, 300 μm × 5 mm, 5 μm granulation, 100 Å.

    • Analytical column: C18, 75 μm × 25 cm, 2 μm granulation, 100 Å; 30 °C.

    • Acetonitrile gradient: from 4% to 60%, in 0.1% formic acid.

    • Flow rate: 300 nl/min (230 min).

     
  2. 2.
    MS and MS/MS conditions:
    • Data-dependent MS/MS mode with survey scans acquired at resolution of 70,000 at m/z 200 in MS mode, and 17,500 at m/z 200 in MS2 mode.

    • Scanning m/z range of 300–2000, positive ion mode.

    • Higher energy collisional dissociation (HCD) ion fragmentation with normalized collision energies set to 25.

     
  3. 3.
    Database search:
    • Perform protein identification using a selected database, e.g., Swiss-Prot human database with a precision tolerance 10 ppm for peptide masses and 0.08 Da for fragment ion masses. Use a selected software for estimation of abundances of identified proteins, e.g., MaxQuant 1.4.1.1 software or Proteome Discover 2.0 for Thermo raw files.

     

4 Notes

  1. 1.

    While maintaining proper dimensions of the chromatographic bed (i.e., height and diameter), a wide range of products can serve as a column for Sepharose 2B. A mini-column suitable for the described conditions can be purchased (i.e., Econo Pac chromatography columns, Bio-Rad) [14]. In order to avoid bed leakage, a frit made of chemically inert material is placed at the outlet of the column. A mini-column is filled with Sepharose 2B, a second frit is placed on the top of Sepharose, and washed 2–3 times with elution buffer (i.e., PBS) until the proper bed height of 10 cm (inner diameter of 1.5 cm) is reached. Bed drying and bed leakage should be avoided.

     
  2. 2.

    We prepare tryptophan stock solution every week and store it at 4 °C; however, freshly prepared solution can also be aliquoted and stored at −20 °C for 3 months. Working solutions (0.1 mgTrp/ml and 0.01 mgTrp/ml in water) should be prepared every day before use.

     
  3. 3.

    Add 40 μl of the Trypsin Resuspension buffer provided along with the enzyme (or 50 mM acetic acid) into the glass vial with enzyme lyophilisate (20 μg), vortex mixture thoroughly, and spin down. Divide the obtained trypsin stock solution into aliquots of 10 μl and store at −20 °C for up to 6 months. Avoid multiple thaw-freeze cycles. Enzymatic activity of trypsin is reversibly blocked only in acidic conditions; therefore, trypsin solutions in water or any digestion buffer (e.g., 25 mM ammonium bicarbonate) should always be used freshly.

     
  4. 4.

    The quality of plastic consumables is of prime importance when samples are to be analyzed by mass spectrometry. Always use materials of the highest quality. We recommend Safe-Lock Tubes and epT.I.P.S.® by Eppendorf AG (Hamburg, Germany). Avoid all kinds of low-bind or siliconized plastics. For long storage of organic solvents borosilicate glass vessels or PTFE-coated plastic bottles are highly recommended instead of polypropylene ones.

     
  5. 5.

    A suitable plastic laboratory box (e.g., 25 cm × 17 cm × 10 cm, L × W × H), equipped with a well-fitting lid, can serve as a humid chamber. Put several sheets of paper towel or cellulose wadding on the bottom of the box and wet them well with water, pour off the excess of water. Place a small tube rack inside. Make sure that the rack does not cover the entire surface of the wetted cellulose. Pre-heat the humid chamber in a laboratory incubator (37 °C). Put a collection tube with a Microcon centrifuge filter in the rack and open the lid of the collection tube. Close the box and leave it in the incubator for 18 h.

     
  6. 6.

    Take as many cells as necessary to achieve proper confluency of cells at the moment of medium collection. We use two 150 cm2 cell culture flasks per an experimental point (the final volume of the conditioned medium is 50 ml). The number of cells is closely related to a cell line, usually we seed between 2 × 106–5 × 106 adherent cells per 150 cm2 in 25 ml of medium. Harvest your cells when they reach 70–80% confluency for adherent cells, or 60–70% of their maximum concentration for cells grown in a suspension.

     
  7. 7.

    Depending on the type of the rotor we suggest: (1) in the case of a swing bucket it is better to collect the supernatant with the use of a Pasteur pipette, since the pellet is localized right on the bottom of the centrifugation tube, it is poorly adherent; (2) in the case of a fixed angle rotor it is better to decant the supernatant in a single motion, since the pellet is localized on a side wall of a tube. This remark is important in subsequent steps of the procedure—in fact it determines the proceeding during the isolation process.

     
  8. 8.

    Directly before use, concentrators are washed with 50 ml of phosphate-buffered saline solution to remove possible postproduction impurities. The solution is then discarded. In order to purify the membrane from glycerine which it is originally covered with, the concentrator is filled with PBS again, centrifuged for 5 min, 700 × g and 4 °C. One should avoid drying out of the membrane. Separation should be performed directly after conditioning of the concentrator.

     
  9. 9.

    Assessment of quantity and quality of exosomes in samples can be performed using five independent methods: (1) Transmission electron microscopy (TEM) techniques: Coat exosomes with 0.125% (w/v) Formvar in chloroform on copper grids. Grids can be stained with 1% (w/v) uranyl acetate in doubly distilled H2O. (2) Protein quantification using a BCA protein assay kit or tryptophan fluorescence measurement. (3) Lipid quantification using a proper reagents kit. (4) Tunable Resistive Pulse Sensing (TRPS) (i.e., qNano by Izon) for size distribution and concentration of particles (according to the manufacturer’s instructions). (5) Western Blot: Perform SDS gel electrophoresis with equal amounts (min. 5–20 μg protein) of each exosomal fraction followed by Western blotting and detection of antigens of interest.

     
  10. 10.
    An exemplary way of preparation of tryptophan standard solutions is presented in Table 1. Although the tryptophan fluorescence method is compatible with many popular solutes employed in protein lysis/extraction buffers (for details refer to [13]), tryptophan standards must reproduce the real sample matrix (i.e., the buffer the assayed proteins/peptides are dissolved in) as accurately as possible.
    Table 1

    Composition of standard solutions of L-tryptophan

    Standard Solution No.

    Buffera

    (μl)

    Trp solution 0.01 μg/μl

    (μl)

    Water

    (μl)

    Total Trp content

    (μg)

    Total protein/peptide content

    (μg)

    Protein/peptide concentration

    (μg/μl)

    1

    80

    0

    20

    0.00

    0.0

    0.000

    2

    80

    4

    16

    0.04

    3.4

    0.034

    3

    80

    12

    8

    0.12

    10.3

    0.103

    4

    80

    20

    0

    0.20

    17.1

    0.171

    Standard Solution No.

    Buffera

    (μl)

    Trp solution 0.10 μg/μl

    (μl)

    Water

    (μl)

    Total Trp content

    (μg)

    Total protein/peptide content

    (μg)

    Protein/peptide concentration

    (μg/μl)

    5

    80

    4

    16

    0.40

    34.0

    0.34

    6

    80

    12

    8

    1.20

    103.0

    1.03

    7

    80

    20

    0

    2.00

    171.0

    1.71

    aexoLB in the case of protein extracts; a mixture of 50 mM Tris–HCl pH 8.5 and water, 1:4 (v/v) in the case of tryptic digests; water in the case of peptide fractions

     
  11. 11.

    Before tryptophan fluorescence measurement the elution buffer in peptide fractions (60% ACN, 0.1% TFA) should be replaced with water, since the presence of acetonitrile and trifluoroacetic acid in such concentrations results in decrease of the method sensitivity by the factor of 4 in comparison to water.

     
  12. 12.

    Cut the lid of a 2 ml reaction tube (two intersecting incisions) and insert a 200 μl pipette tip in the obtained slit, press firmly. Cut six plugs of a strong anion exchanger (SAX) extraction filter with the use of a blunt needle, transfer the needle to the pipette tip and push the plugs out of the needle with the use of a plastic or metal wire. Press the material tightly in the tip—the plugs should not separate one from another when in use, nevertheless too tight packing may block a tip column. Repeat the procedure with C18 extraction filter, but use three plugs instead of 6.

     
  13. 13.

    Avoid contamination during sample preparation. Always use gloves and proper lab coats. It is easy to contaminate a sample, e.g., with keratins or other abundant proteins when touching lab equipment or sample tubes without gloves. When using gloves be careful not to touch things like door handles, computer mouse or keyboard etc. If still keratins are overrepresented in an identification report (MASCOT), check for other contamination sources like dust in the lab and prepare your samples under a hood. Other contamination sources are polyethylene glycols (PEGs) and plasticizers like phthalate derivatives. Use only high quality plastics or glass (see Note 4 ). PEGs are often introduced when using detergents in your lab.

     
  14. 14.

    For peptides separation using LC/MS, different column configurations may be alternatively adapted. The best configuration is as described here, using a pre-column (trap column) prior to the proper separation step, but also direct column separation may be applied. It should be noted here that total capacity of a column and a pre-column should be investigated to avoid their overloading.

     
  15. 15.
    This procedure can also be adapted to peptide labeling approaches like iTRAQ technique instead of the described label-free approach. If applicable, several remarks should be considered:
    1. (a)

      Not all MS systems are compatible with peptide isobaric labeling on MS2, for example, ion trap mass spectrometers are limited in registration of all fragment ions, reporter ions may be missed in this case.

       
    2. (b)

      When using QExactive instrument remember to set up “first mass” measurement at 100 m/z for acquisition of all MS2 spectra.

       
    3. (c)

      When using labeling at MS stage (i.e., SILAC, ICAT), remember that sample complexity is doubled which may cause sensitivity or suppression problems.

       
     
  16. 16.

    This procedure may also be adapted to protein modifications analysis (PTMs). Just remember to include proper modifications and their sites as variable modifications in MASCOT search parameters. Avoid searching too many modifications at a time as it can give rise to unspecific peptide identification. When dealing with modifications occurring at lysine (or arginine) residues consider increasing the number of possible miscleavages.

     
  17. 17.

    In a label-free approach, samples from a given experiment should be analyzed in one batch when possible (in a randomized order). Proper calibration of a mass spectrometer should be performed before the first analysis and after every 10 or 15 samples. If applicable, use quality control samples (QC) between selected runs. All these remarks are important for proper further data formatting and preparation prior to statistical analysis (data normalization and alignment).

     
  18. 18.
    Load internal syringe of the fraction collector with a matrix solution consisting of:
    1. (a)
      For nano-LC fractions:
      • 748 μl of 95% ACN in water, 0.1% TFA.

      • 36 μl of HCCA saturated in 90% ACN in water, 0.1% TFA.

      • 8 μl of 10% trifluoroacetic acid in water.

      • 8 μl 100 mM NH4H2PO4 in water.

       
    2. (b)
      For external calibrants:
      • 748 μl of 85% ACN in water, 0.1% TFA.

      • 36 μl of HCCA saturated in 90% ACN in water, 0.1% TFA.

      • 8 μl 10% trifluoroacetic acid in water.

      • 8 μl 100 mM NH4H2PO4 in water.

       
     

Notes

Acknowledgements

This work was supported by the National Science Centre, Poland, Grant 2013/11/B/NZ7/01512.

References

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Monika Pietrowska
    • 1
  • Sonja Funk
    • 2
    • 3
  • Marta Gawin
    • 1
  • Łukasz Marczak
    • 4
  • Agata Abramowicz
    • 1
  • Piotr Widłak
    • 1
  • Theresa Whiteside
    • 2
  1. 1.Center for Translational Research and Molecular Biology of CancerMaria Sklodowska—Curie Memorial Cancer Center and Institute of Oncology, Gliwice BranchGliwicePoland
  2. 2.Department of PathologyUniversity of PittsburghPittsburghUSA
  3. 3.Department of OtolaryngologyUniversity of Duisburg-EssenEssenGermany
  4. 4.Institute of Bioorganic ChemistryPolish Academy of SciencesPoznańPoland

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