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Protein‐Protein Interactions Identified by Pull‐Down Experiments and Mass Spectrometry

互联网2013-12-31

2707

Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

The aim of this unit is to provide a method for the identification of new protein?protein interactions. Pull?down experiments with GST fusion proteins attached to glutathione beads are a screening technique for identification of protein?protein interactions. When coupled with mass spectrometry, pull?downs can be considered as the protein?based equivalent of a yeast two?hybrid screen. To improve the isolation of specific binding partners, pull?down methods are described involving the use of cross?linking, large?scale tissue lysates, and spin columns. Alternative techniques are detailed for isolating activation state?dependent protein interactions with small GTPases. Appropriate methods of sample preparation for mass spectrometry?based identification of interacting proteins are described, including specialized gel staining techniques, band excision, and in?gel tryptic digestion. Data interpretation and the most commonly encountered problems are discussed.

Keywords: Pull?down; protein?protein interactions; small GTPase; GST fusion protein; mass spectrometry; spin column; cross?linking; in?gel digestion; tissue lysate

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Strategic Planning
Basic Protocol 1: The Pull‐Down Experiment
Alternate Protocol 1: Effector Isolation with GST‐Tagged Small Gtpases
Support Protocol 1: GST Fusion Protein Purification
Support Protocol 2: Cross‐Linking GST Fusion Proteins to GSH‐Agarose
Support Protocol 3: Preparation of Large‐Scale Tissue Lysates
Basic Protocol 2: Sample Preparation for Mass Spectrometry
Support Protocol 4: Recycling of GSH Agarose
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: The Pull‐Down Experiment

Materials

Tissue lysate (see protocol 5 )
Bead storage buffer: 20 mM Tris·Cl, pH 7.4 ( appendix 2A )/50% (v/v) glycerol
GSH‐agarose beads (e.g., glutathione‐Sepharose 4B; Amersham Biosciences)
Wash buffers 1 or 2, and 3 (see reciperecipes )
1× SDS sample buffer (unit 6.1 )
Benchtop centrifuge, 4°C
Appropriately sized tubes that can be centrifuged
End‐over‐end rotator
Empty plastic column (e.g., Poly‐Prep from Bio‐Rad; to suit lysate volume) or Miracloth (Calbiochem)
MicroSpin columns (Amersham Biosciences), or other spin columns (see Table 17.5.1 )
85°C water bath

Additional reagents and equipment for preparing GST fusion proteins attached to GSH‐agarose beads (see protocol 3 ), cross‐linking of fusion proteins (optional; see protocol 4 ), preparing tissue lysate (see protocol 5 ), and SDS‐PAGE (unit 6.1 )

Alternate Protocol 1: Effector Isolation with GST‐Tagged Small Gtpases

1 M MgCl₂ ( appendix 2A )
Bead storage buffer (20 mM Tris·Cl, pH 7.4 ( appendix 2A )/50% (v/v) glycerol) containing 2.5 mM MgCl₂ (added from 1 M MgCl₂ stock; appendix 2A )
Small GTPase loading buffer (see recipe )
100 mM GDP and GTP (or GTPγS) stock solutions (prepare fresh on the day of the experiment and keep cold):
- 100 mM GDP (mol. wt. 463.2) = 1 mg in 21.6 µl H₂ O
- 100 mM GTP (mol. wt. 567.1) = 1 mg in 17.6 µl H₂ O
- 100 mM GTPγS (mol. wt. 627.2) = 1 mg in 15.9 µl H₂ O
Wash buffers 4 or 5, and 6 (see reciperecipes )

Support Protocol 1: GST Fusion Protein Purification

Materials

Glycerol stock of E. coli cells containing GST fusion protein expression vector (see appendix 3A and Coligan et al., )
LB medium and LB agar plates (see appendix 2A ) containing 100 µg/ml ampicillin (or other appropriate antibiotic selection for GST expression vector used)
100 mM isopropyl‐1‐thio‐β‐D‐galactoside (IPTG), filter sterilized
Bleach (e.g., Clorox)
Bacterial lysis buffer: 20 mM Tris·Cl, pH 7.4 ( appendix 2A )/250 mM NaCl
100 mg/ml lysozyme
100 mM PMSF ( appendix 2A )
20 mg/ml leupeptin
Liquid nitrogen and appropriate storage canister
1 mg/ml DNase I
10% (v/v) Triton X‐100 ( appendix 2A )
Bacterial lysis buffer (see above) containing 1% (v/v) Triton X‐100
1% (w/v) SDS
Wash buffer 1 (see recipe )
Bead storage buffer: 20 mM Tris·Cl, pH 7.4 ( appendix 2A )/50% (v/v) glycerol
1× and 3× SDS sample buffer (unit 6.1 )
Bovine serum albumin (BSA; optional)

Shaking bacterial incubator
Culture tubes, sterile
1‐liter conical flasks (Pyrex or autoclavable plastic), sterilized by autoclaving or baking
Bugstoppers (Whatman), sterilized by autoclaving, or other sterile, gas‐permeable flask closures
Probe sonicator (e.g., Branson)
Refrigerated high‐speed centrifuge and benchtop centrifuge
15‐ml and 50‐ml polypropylene screw‐cap tubes (e.g., Falcon)
Pipet tip cut to increase opening to >1 mm
85°C water bath
MicroSpin columns (Amersham Biosciences)

Additional reagents and equipment for growing bacterial cells (see appendix 3A ), SDS‐PAGE (unit 6.1 ), Coomassie blue staining of gels (unit 6.6 )

Support Protocol 2: Cross‐Linking GST Fusion Proteins to GSH‐Agarose

Materials

GST fusion protein beads (prepared as in protocol 3 , with the protein concentration determined)
PBS ( appendix 2A )
Disuccinimidyl suberate (DSS), 13.2 mg in screw‐cap tube (see recipe )
bis(sulfosuccinimidyl) suberate (BS3; see recipe )
Dimethylsulfoxide (DMSO; H₂ O‐free, i.e., fresh)
Quenching buffer: 1 M Tris·Cl, pH 8.2 ( appendix 2A ), room temperature
Glutathione wash buffer (see recipe ), room temperature
Wash buffer 1 (see recipe ), ice‐cold
Cross‐linking wash buffer: 25 mM Tris·Cl, pH 7.6 ( appendix 2A )/1.5 M NaCl, ice‐cold
Bead storage buffer: 20 mM Tris·Cl, pH 7.4 ( appendix 2A )/50% (v/v) glycerol
Appropriately sized columns (e.g., Bio‐Rad 2‐ml Bio‐Spin columns or 10‐ml Poly‐Prep columns)

Support Protocol 3: Preparation of Large‐Scale Tissue Lysates

Materials

200 g sheep brain (see recipe )
Buffers A, B, C, and D (see reciperecipes ; prepare in advance and cool overnight to 4°C)
GSH‐agarose beads (e.g., glutathione‐Sepharose 4B; Amersham Biosciences)
10% (v/v) Triton X‐100

Colander or tea strainer
1‐liter plastic beakers
Ultra‐Turrax tissue homogenizer (IKA Labortechnik) with 25‐mm‐diameter blade/tip
Refrigerated centrifuge and 0.5‐liter centrifuge bottles
Miracloth (Calbiochem)
50‐ml screw‐cap plastic tubes

NOTE: Sometimes precipitates accumulate during freeze/thaw of these samples, and this is particularly true for the peripheral membrane extract (see note above). All frozen aliquots must be centrifuged after defrosting at 35,000 × g (or maximum speed) for 30 min at 4°C, before incubation with recombinant GST fusion protein beads. It is essential to clear any debris before adding the extract to the beads. It may also be useful to pre‐clear the cytosol extracts a third time before use, to remove any remaining traces of endogenous GST (this is optional; see protocol 1 or the protocol 2Alternate Protocol ). All volumes of beads in this protocol refer to bed volumes.

Basic Protocol 2: Sample Preparation for Mass Spectrometry

Materials

Unstained SDS acrylamide gel ( protocol 1 or protocol 2Alternate Protocol )
Colloidal Coomassie stain (see recipe )
1% (v/v) acetic acid
Zinc stain solutions I and II (see reciperecipes )
50% (v/v) and 100% acetonitrile
25 and 50 mM ammonium bicarbonate (prepare from 1 M stock; see recipe )
25 mM ammonium bicarbonate in 50% (v/v) acetonitrile (prepare from 1 M ammonium bicarbonate stock; see recipe )
Zinc destain solution (see recipe )
Digestion buffer (see recipe ), ice‐cold
5% (v/v) formic acid
50% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid (TFA)
Sealable plastic containers of appropriate size for gel
Platform shaker
Flatbed scanner (preferably with transparency adapter for Coomassie stain)
Computer running graphics software, e.g., Adobe Photoshop
Light box
Scalpel (with new blades) or fine‐gauge needles
Siliconized, hydrophobically coated, or highly polished plastic microcentrifuge tubes (e.g., Axygen Maxymum recovery clear 0.6‐ml tubes), pipet tips (Axygen Maxymum recovery pipet tips), and gel‐loading tips (e.g., Sorenson Multi Miniflex round sterile gel loading pipet tips, 0.1 to 10 µl)
Stainless steel mortar and pestle (e.g., Quantum Scientific)
Heat‐sealable plastic sleeves and heat sealer
Pieces of cardboard and large sturdy flat box to accommodate gels
50‐ml screw‐cap tubes
Heavy‐gauge needle
Thermomixer/shaker (to fit microcentrifuge tubes)
Bath sonicator

Support Protocol 4: Recycling of GSH Agarose

Materials

Beads for recycling (from pre‐clearing steps in protocol 1 , the protocol 2Alternate Protocol , or protocol 5 )
Glutathione wash buffer (see recipe ), room temperature
Bead recycling buffer: 100 mM glycine hydrochloride, pH 2.3, room temperature
Phosphate buffered saline (PBS; appendix 2A ), room temperature
20% (v/v) ethanol
1% (w/v) SDS (see appendix 2A )
0.1% (v/v) Triton X‐100 (see appendix 2A )
End‐over‐end rotator

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Figures

Figure 17.5.1 Flowchart for strategic design and the experimental processes described in this unit, showing how the planning and analysis stages (left‐hand side; a summary of the Strategic Planning section) are linked with the experimental protocols (right‐hand side). It is also a reference for how the protocols relate to one another.

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Figure 17.5.2 The spin column–based pull‐down experiment. An illustrative description of the approach taken when an entire pull‐down experiment is performed within the spin column. This differs from the protocol presented in and the Alternate Protocol, as it is designed for smaller‐scale experiments. The large‐scale experiments in the text intersect with this figure at the point marked with an asterisk ~undefined), corresponding to step in and the Alternate Protocol.

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Figure 17.5.3 Schematic of describing the in‐gel digestion technique and peptide extraction steps. For graphical reasons, the gel pieces are depicted as whole (to show the state of the stain), but would in practice be diced. Refer to the main body of the text for more details. Abbreviation: AMBIC, ammonium bicarbonate, pH 8.0.

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Figure 17.5.4 Regeneration of GSH agarose. The expected results from are shown. Lane 1 shows the stained endogenous GST proteins present bound to a batch of GSH agarose that has been used for preclearing a tissue lysate. Lanes 2 to 4 show the sequential treatments which remove protein from the beads, and enable their reuse for preclearing procedures.

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Figure 17.5.5 Sample results from a GST‐RalA pull‐down experiment. Shown is a Coomassie Blue–stained gel of a pull‐down from sheep brain cytosol using GST‐tagged RalA. For a full discussion, see Anticipated Results. Stained protein bands that have been identified by MALDI‐TOF MS are indicated on the right. Features marked with an asterisk ~undefined) are interfering species. Bands that have been identified as erroneously migrating GST‐RalA are marked G. The bands marked N are either nonspecific sheep proteins, fragments of GST‐RalA, copurified bacterial contaminants, or real nucleotide‐independent binding proteins.

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Videos

Literature Cited

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