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Using mass spectrometry for protein-protein interaction studies. Katalin Medzihradszky PC 204. Identifying interacting partners. bait- fish-(fractionate)-digest-identify * immunoprecipitation * affinity chromatography with a protein * TAP-TAG purification

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Using mass spectrometry for protein protein interaction studies

Using mass spectrometry for protein-protein interaction studies

Katalin Medzihradszky

PC 204

Identifying interacting partners
Identifying interacting partners studies

  • bait- fish-(fractionate)-digest-identify

    * immunoprecipitation

    * affinity chromatography with a protein

    * TAP-TAG purification

  • by cross-linking first then bait and fish ect.

Specific handles
Specific “handles” studies

  • Immunprecipitation

  • His-tag

  • biotin

  • GST

  • FLAG-tag

  • and the combination thereof


Köcher & Superti-Furga NATURE METHODS studies| VOL.4 NO.10 | OCTOBER 2007

Tandem Affinity Purification technology studies

  •  Optimized method to purify protein complexes with high efficiency and specificity by the sequential use of two tags

  • Minimum of contamination of extraneous proteins

Johannes Graumann et al, “ studiesApplicability of Tandem Affinity Purification MudPIT to Pathway Proteomics in Yeast” Mol. Cell. Proteomics, Mar 2004; 3: 226 - 237.

Schematic representation of the HPM tag.

Other examples
Other examples studies

  • Dennis D. Wykoff and Erin K. O’SheaIdentification of Sumoylated Proteins by Systematic Immunoprecipitation of the Budding Yeast ProteomeMol. Cell. Proteomics, Jan 2005; 4: 73 - 83.

  • Anna Shevchenko, Daniel Schaft, Assen Roguev, W. W. M. Pim Pijnappel, A. Francis Stewart, and Andrej ShevchenkoDeciphering Protein Complexes and Protein Interaction Networks by Tandem Affinity Purification and Mass Spectrometry: Analytical PerspectiveMol. Cell. Proteomics, Mar 2002; 1: 204 - 212.

  • Silvia A. Synowsky, Robert H. H. van den Heuvel, Shabaz Mohammed, W. W. M. Pim Pijnappel, and Albert J. R. HeckProbing Genuine Strong Interactions and Post-translational Modifications in the Heterogeneous Yeast Exosome Protein ComplexMol. Cell. Proteomics, Sep 2006; 5: 1581 - 1592.

  • Pierre-Olivier Angrand, Inmaculada Segura, Pamela Völkel, Sonja Ghidelli, Rebecca Terry, Miro Brajenovic, Kristina Vintersten, Rüdiger Klein, Giulio Superti-Furga, Gerard Drewes, Bernhard Kuster, Tewis Bouwmeester, and Amparo Acker-PalmerTransgenic mouse proteomics identifies new 14-3-3 associated proteins involved in cytoskeletal rearrangements and cell signaling

    Mol. Cell. Proteomics, Sep 2006; doi:10.1074/mcp.M600147

2d elfo differently proteasome complex of drosophila melanogaster
2D studies-elfo differentlyProteasome complex of Drosophila melanogaster





Pros35 10/14 (71%) 33%

Pros7 34/36 (94%) 63%

Pros6 12/12 (100%) 42%

Pros28.1 14/21 (66%) 54%

Pros29 19/23 (83%) 59%

Pros2 13/15 (86%) 42%

GC12000 13/14 (93%) 35%

ProsMA5 22/44 (50%) 56%

Pros25 12/15 (80%) 50%

Pros3 18/24 (75%) 53%

Pros26 6/15 (40%) 46%

Pros5 11/12 (92%) 26%

GC17331 11/23 (47%) 46%

l(2)05070 15/19 (79%) 72%

Sümegi M, Hunyadi-Gulyás E, Medzihradszky KF, Udvardy A.

Biochem Biophys Res Commun. 2003 Dec 26;312(4):1284-9.

Catch of the day
Catch of the day studies

  • the protein of interest

  • its interactive partners – complexes; tight interactions

    What else is in the bag?

The usual suspects
the usual suspects studies

There is always some nonspecific interaction

and there are a lot of sticky proteins

Prevention strategy?

Wild type studies

Tagged protein



Mixing prior to the


MS analysis

J Proteome Res. 2005 Sep-Oct;4(5):1752-6.

Tackett AJ, DeGrasse JA, Sekedat MD, Oeffinger M, Rout MP, Chait BT.

Other biological contamination
Other “biological contamination” studies

  • Human keratin (in-gel digestions)

  • BSA – from medium, from enzyme preps…

  • All kind of proteins from the expression systems

  • Avidin, GST-bait protein – from the appropriate affinity columns

  • IgG – from immunprecipitations, affinity fishing

  • Rubber tree proteins – from latex gloves

  • Caseins – from using the same dish for Western and staining (in-gel digestions)

What about weak temporary interactions
What about weak/temporary interactions? studies

Cross-linking strategies

* Formaldehyde – cheap, reversible, can be performed in vivo

Schmitt-Ulms G, et al, Time-controlled transcardiac perfusion cross-linking for the study of protein interactions in complex tissues.Nat Biotechnol. 2004 Jun;22(6):724-31. Epub 2004 May 16.

Cortnie Guerrero et al., An Integrated Mass Spectrometry-based Proteomic Approach: Quantitative Analysis of Tandem Affinity-purified in vivo Cross-linked Protein Complexes (qtax) to Decipher the 26 s Proteasome-interacting Network Mol. Cell. Proteomics, Feb 2006; 5: 366 - 378.

Chemical Cross-linking of Proteins by Formaldehyde studies

Orlando V, et. al. Methods. 1997;11(2):205-14.

Schematic Diagram of an Integrated Proteomics Approach studies

to Study the 26S Proteasome Interacting Proteins












1.Freeze interaction

(in vivo cross-linking)


2.Tandem Affinity



3. Enzymatic Digestion

5. Database





Protein Identification

Guerrero C, Tagwerker C, Kaiser P, Huang L. Mol Cell Proteomics. 2006; 5(2):366-78.

Christian Tagwerker et al, studiesA Tandem Affinity Tag for Two-step Purification under Fully Denaturing Conditions: Application in Ubiquitin Profiling and Protein Complex Identification Combined with in vivoCross-Linking Mol. Cell. Proteomics, Apr 2006; 5: 737 - 748.

A tandem affinity tag consisting of a His6 element and a signal sequence for in vivo biotinylation.

on-bead digestion

Standard silac protocol
Standard SILAC Protocol studies

Purification After Mix (PAM)

HTBH only


12C6-Arg/12C6 -Lys


Cell Lysis

Mixing Equal Amounts of Lysates

Affinity Purification

TCA Precipitation

LysC/Trypsin Digestion


Protein Identification and Quantitation


Time controlled tc pam silac
Time-Controlled (Tc) –PAM-SILAC studies

HTBH only




20 min

Cell Lysis

Mixing Equal Amounts of Lysates

1 hr

Time-controlled Incubation

Affinity Purification

TCA Precipitation

2 hr

LysC/Trypsin Digestion


Protein Identification and Quantitation


Modified SILAC Protocol studies

Mix After Purification (MAP)

HTBH only



13C615N4-Arg/13C615N2 -Lys

Cell Lysis

Affinity Purification of Equal Amount of Cell Lysates Separately

Mixing of Purified Complexes

TCA Precipitation

LysC/Trypsin Digestion


Protein Identification and Quantitation


HTBH studies




Strategies for Quantifying Protein Interactions of Protein Complexes

Mass Spectrometric Analysis

Incubation Time




SILAC Labeling









20 min





2 hr



Wang & Huang

IMol. Cell. Proteomics, 7: 46 – 57 (2008).






Characterizing known complexes
Characterizing known complexes studies

  • Identifying interacting surfaces by covalent crosslinking and MS

  • Identifying interacting surfaces by H/D exchange

  • MS and MS/MS of the complexes

Crosslinking Chemistry for Protein Complex Analysis studies

NHS-ester crosslinkers can undergo both Aminolysis and Hydrolysis



  • Fundamental Problem of Crosslinking Large studies

  • Protein Complexes/Molecular Machines:

  • True Intermolecular Crosslinks are formed in very low yield.

  • Reaction mixture consists largely of unmodified and

  • “dead-end”-modified proteins.

  • Enrichment scheme is necessary if we are to

  • apply crosslinking on a large scale.

Mike Trnka

How to specifically enrich intermolecular studies

(and intramolecular) xlinked peptides.

Affinity Handle/Biotin

Cleavage Site

+Protein Complex

Amine-Reactive Grps

Dead-end Crosslinks should leave a chemical handle: studies

Type 1,2 Xlink



Type 0 (“dead-end”) Xlink

Non-cleavable Affinity Tag/Biotin

How to enrich intermolecular (and intramolecular) studies

xlinked peptides.

Type 1,2 Xlink


Type 0 (“dead-end”) Xlink

Bind to Streptavidin and Cleave: studies

Type 1,2 Xlink


Type 0 (“dead-end”) Xlink

ICAT-XL, 1 studiesst generation affinity crosslinker:

Chu, F. et al.JACS128, 10362-3 (2006).

Without enrichment studies

Proc Natl Acad Sci U S A. 2004 Nov 23;101(47):16454-9. Epub 2004 Nov 16.

Unraveling the interface of signal recognition particle and its receptor by using chemical cross-linking and tandem mass spectrometry.

Chu F, Shan SO, Moustakas DT, Alber F, Egea PF, Stroud RM, Walter P, Burlingame AL.

Cross-linking of E. coli Ffh·FtsY complex. (A) SDS/PAGE analysis of the cross-linking reaction. (B and C) Total ion chromatograms (TIC) of the tryptic digestion mixture of the cross-linked Ffh·FtsY complex (B) and the control (C). (Insets) The mass spectra of the peptides eluted at ≈42.5 min.

T. aquaticus studies

Ffh NG

T. aquaticus






















SRP complex w/ receptor



Model Overlaid w/ Xtal Struct.

Domain Rearrangement



Chu, F. et al.PNAS101, 16454-9 (2004).

Protein Prospector will help! studies

Mol Cell Proteomics. 2010 Jan;9(1):25-31. Epub 2009 Oct 6.

Finding chimeras: a bioinformatics strategy for identification of cross-linked peptides.

Chu F, Baker PR, Burlingame AL, Chalkley RJ.

Identification of the same cross-linked species from an ecotin dimer, using 2 different linkers

Application of amide proton exchange mass spectrometry for the study of protein-protein interactions.

Mandell JG, Baerga-Ortiz A, Croy CH, Falick AM, Komives EA.

Curr Protoc Protein Sci. 2005 Jun;Chapter 20:Unit20.9.

  • Flow chart diagram of the two types of amide proton exchange experiments used to study protein-protein interfaces.

  • In the on-exchange experiment, the protein-protein complex shows a region in which less deuterium is incorporated when compared with control samples in which each protein is deuterated separately.

  • In the off-exchange experiment, each protein is allowed to incorporate deuterium separately, the deuterated proteins are allowed to complex, and then deuterium atoms are off-exchanged with hydrogen atoms by dilution in H2O; residues located at the protein-protein interface are characterized by the retention of deuterons in the protein-protein complex as compared with a lack of retention in control experiments in which only one of the two proteins is present.

Example for on-exchange experiments used to study protein-protein interfaces.

  • Example of a peptide mass envelope resulting from pepsin digestion of (i) nondeuterated CheB protein; (ii) a deuterated protein complex involving CheB; and (iii) CheB alone after being subjected to the same deuteration period as the complex in (ii). The mass envelope broadens somewhat upon deuteration, and there is less deuteration of the peptide in the protein-protein complex than in the protein alone.

  • Graph of number of deuterons incorporated into the peptide shown in panel A versus deuteration time. Circles, uncomplexed CheB; squares, complexed CheB.

Example for off-exchange experiments used to study protein-protein interfaces.

(A) Nondeuterated peptide fragment from the uncomplexed protein.

(B) Peptide fragment from the uncomplexed protein after on-exchange for 10 min.

(C) Peptide fragment from the uncomplexed protein after on-exchange for 10 min followed by off-exchange for 10 min. Some residual deuteration is seen, due to the fact that some D2O remains in the H2O buffer used for off-exchange.

(D) Peptide fragment from the complexed protein after on-exchange (performed on each component of the complex separately) for 10 min followed by off-exchange (performed on the bound complex) for 10 min. In the bound complex, the peptide retains deuterium throughout off-exchange, whereas in the uncomplexed state, it does not. This indicates that the peptide is probably part of the protein-protein interface region.

What can we learn from H/D exchange? experiments used to study protein-protein interfaces.

Structure of PPACK thrombin with loops colored according to the change in amide H/2H exchange seen upon activation of thrombin. The catalytic triad residues (H57CT, D102CT, and S195CT) are shown as sticks and in green. Loops in red become more dynamic upon thrombin activation. Loops in blue become less dynamic upon thrombin activation.

PPACK thrombin =(D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone- treated alpha-thrombin)

Its X-ray structure was determined

Amide H/2H exchange reveals a mechanism of thrombin activation.

Koeppe JR, Komives EA. Biochemistry. 2006 Jun 27;45(25):7724-32.

Chem Rev. 2007 Aug;107(8):3544-67. Epub 2007 Jul 25. experiments used to study protein-protein interfaces.

Protein complexes in the gas phase: technology for structural genomics and proteomics.

Benesch JL, Ruotolo BT, Simmons DA, Robinson CV.

Mass spectrometry is used at every level experiments used to study protein-protein interfaces.

Pyramid of protein organization states.

Conventional and nanoelectrospray MS of a experiments used to study protein-protein interfaces. GroEL complex complex.

The nESI spectrum displays a series of peaks around 11500 m/z which correspond to the 800 kDa tetradecamer.

Conventional ESI of the same solution results in poorly resolved “humps” centered on 12500, 16000, and 18500 m/z, identified as the tetradecamer, a dimer of tetradecamers, and a trimer of tetradecamers, respectively. There is also a signal at low m/z which corresponds to the GroEL monomer. The charge state distribution is broader and bimodal, and the formation of nonspecific oligomers is increased. These results indicate some of the benefits of using nESI.

A smaller initial droplet size leads to less nonspecific aggregation (both protein−protein and protein−salt), and the gentler interface conditions possible, while still allowing adequate desolvation, lead to less dissociation and disruption of oligomeric structure.

Solution conditions were 200 mM ammonium acetate, pH 6.9, and a protein concentration of 2 μM tetradecamer. Spectra were obtained on a modified Q-ToF 2 (Waters/Micromass).


normal ESI

Nonspecific aggregation during ion formation by nESI. experiments used to study protein-protein interfaces.

Initial droplets undergo asymmetric fission, offspring droplets containing none, one, or several of the molecules of interest are formed

(A). These droplets are on the order of 18 nm.50 A large proportion of the droplets formed are vacant, but at higher concentrations, more occupied droplets are formed

(B). The relative proportions of the droplets which contained none, one, two, and three molecules vary according to the concentration. Droplets containing multiple copies of proteins eventually will lead to the formation of nonspecific aggregates. (simulation; charged residue model)

Measured values @ different V experiments used to study protein-protein interfaces. acc

Theoretical mass

Adduction of solvent molecules and buffer ions to proteins.

(A) The theoretical deconvoluted spectrum of a pure protein complex would appear at a mass representing the primary sequence, with a peak width defined by the isotopic distribution and instrumental resolution

The masses of protein complexes observed are higher

(B) Examination of the 68+ charge state of GroEL at different activation voltages.

The amount of accelerating voltage (low to high) required to achieve the indicated peak width is indicated by the color of the points in panel B.

A Q-ToF type instrument customized for the transmission and analysis of protein complexes.

(A) This analyzer is operates at a reduced RF frequency, allowing the selection and transmission of high m/z ions. The dissociation of protein complex ions requires higher collision cell gas pressures and accelerating voltages relative to “normal” parameters.

(B) Improved transmission is achieved by adjusting the velocity of the ions, via altering the pressures in the instrument.

C). As the pressure is increased from 9.3 μbar, the total signal intensity (width of the bars) increases. Examining how the ion signal divides between the four segments of the MCP plate suggests that, at pressures in the source region below and above the optimum, ions over- and undershoot the detector, respectively.

Number of collisions experienced and time spent in the collision cell.

Upper panel:

the calculated number of collisions relative to mass, by cytochrome c monomer (violet), transthyretin tetramer (blue), MjHSP16.5 24mer (green), GroEL tetradecamer (orange), and the 70S ribosome from Thermus thermophilus (red).

The inset shows the linear dependence of the number of collisions on pressure for MjHSP16.5.

Lower panel:

Time spent in the collision cell vs. mass.

The inset shows the accelerating voltage and pressure dependency of this time (for MjHSP16.5, 47+ charge state).

These calculations are based on a collision cell length of 18.5 cm, a gas pressure of 30 μbar argon, and an accelerating voltage of 200 V.

Dependence on mass @ constant Ar pressure collision cell.

Energy conversion during collisional activation.

Upper panel

Simulations conversion of energy from kinetic to internal modes

cytochrome c monomer (violet)

transthyretin tetramer (blue)

MjHSP16.5 24mer (green)

GroEL tetradecamer (orange)

70S ribosome from Thermus thermophilus (red)

Middle panel

The effect of varying pressure on this energy conversion process for a single species, MjHSP16.5. Different pressures (labeled from 50 μbar, violet, to 10 μbar, red) of argon are simulated.

Lower panel

The effect of the mass of the gas (the noble gases from radon, violet, to neon, red) (-using the same ion as above)

Inset into these panels are the dependencies for 50% conversion.

These simulations show that in order for sufficient conversion of kinetic energy into internal modes to occur, in this length of collision cell, the use of higher pressures and/or heavier target gas is preferable.

Dissociation pathway of a multiprotein complex. collision cell.

(A) CID of the 32+ charge state of the TaHSP16.9 dodecamer (violet) results in the formation of complementary monomers at low m/z and 11mers at high m/z (both blue). At higher accelerating voltages (>100 V), a second distribution of monomers as well as decamers is observed (both cyan). This is indicative of a sequential dissociation reaction (white arrows).

(B) Plotting the relative abundance of the different oligomeric species as a function of accelerating voltage

(C) How long it takes the protein-complexes to dissociate

Experiments were performed on a modified Q-ToF 2.105 The solution of TaHSP16.9 was infused by nESI at a concentration of 1.4 μM in 200 mM ammonium acetate, pH 6.9.14

Applications of collisional activation to the study of protein complexes.

Information as to oligomeric composition (green) can be obtained from the identity of dissociation products (purple), as well as from beneficial effects regarding the parent ion (pink).

A detailed examination of the reaction pathway can reveal certain parameters (blue) which can allow the determination of interaction strengths (red) and information as to oligomeric organization and size (orange).

PNAS protein complexes.

Proceedings of the National Academy of Sciences of the United States of America

Quaternary dynamics and plasticity underlie small heat shock protein chaperone function

1. Florian Stengela,

2. Andrew J. Baldwinb,

3. Alexander J. Paintera,

4. Nomalie Jayac,

5. Eman Bashac,

6. Lewis E. Kayb,

7. Elizabeth Vierlingc,

8. Carol V. Robinsona,1, and

9. Justin L. P. Benescha,1

Temperature-induced changes in HSP18.1 oligomerization. protein complexes.

At the lowest temperatures HSP18.1 exists almost exclusively as a 216 kDa dodecamer, with charge states centered around 6,350 m/z

At higher temperatures monomers and dimers are observed at low m/z, and higher-order oligomers at high m/z, demonstrating the temperature-dependent dissociation and augmentation of the dodecamers.

B A plot of the relative amount of HSP18.1 subunits existing in different oligomeric states.

C Close examination of the abundance of the different species at 46° C shows a clear preference relative to a Gaussian distribution (p < 0.01) for oligomers with an even number of subunits.

D Plots of the difference in free energy between a subunit free in solution or incorporated into either a dimer or dodecamer follow linear trends, suggesting that neither undergo significant structural change upon thermal activation. In all graphs the data and error bars represent the mean and standard deviation of three independent experiments.

Time-course of the formation of complexes between HSP18.1 and unfolding Luc. ( Luc=firefly luciferase, extremely thermo-sensitive )

B Quantifying the relative abundances of the different species shows the initial rapid binding of Luc by HSP18.1, followed by further incorporation of HSP18.1 into the resultant complexes. The average mass of these complexes (Red, right-hand y-axis) mirrors this behavior, revealing the presence of distinct “binding” and “augmentation” steps in the chaperone action of HSP18.1.

Identification and quantification of HSP18.1:Luc complexes by means of tandem-MS.

A Spectrum of a complex formed between HSP18.1 and Luc at a 1:0.1 ratio. Unbound dodecamer is observed around 6,350 m/z, and a broad area of signal, characteristic of a polydisperse ensemble, is observed above 8,000 m/z.

B Selection of ions at a particular m/z, gray, and activation of selected ions leads to the removal of highly charged HSP18.1 subunits (≈1,800 m/z) from the parent oligomers. Complementary stripped complexes appear at high m/z.

C Relative quantification of the different complexes from the heights of the peaks reveals a number of different stoichiometries of HSP18.1 bound to one Luc, and a dominance (p < 0.01) of those containing an even number of HSP18.1 subunits.

A by means of tandem-MS. The dodecameric form of HSP18.1 represents a “storage” form, which is in equilibrium with suboligomeric species, and higher-order oligomers. These higher-order species are themselves continually recycling through the loss and reincorporation of dimers and monomers. Upon heat-shock the equilibria shift (Red) from the dodecamer to dissociated species, and unfolding clients are bound to form sHSP:client complexes.

B From the number of different species we observe here for the different states of HSP18.1 (Orange) we can extrapolate to systems incorporating multiple sHSPs (Bars). Based on the ability of related sHSPs to hetero-oligomerise, the number of potential species (NComb) can be calculated using the inset equation, where NsHSP is the number of compatible sHSPs, i the number of HSP18.1 subunits in the oligomer, and NClient the number of different bound states for a particular i. This reveals a remarkably extensive potential sHSP network, presumably catering for the array of different unfolding clients requiring protection from aggregation during cellular stress.

Conclusion for small HSP-containing complexes