Nagarjuna Nagaraj MPI of Biochemistry, Core Facility Mass Spectrometry – nagaraj@biochem.mpg.de

03.12.2015

MS core facility at MPI Biochemie

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People and infrastructure Lissy Vicky Naga Nicole

• TOF(time of flight) – ESI-TOF – ESI-qTOF (high resolution TOF)

• Orbitrap – 2 X QExactiveHF bench-top machines

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MS core facility MPI-Biochemie

Top-down analysis

Bottom-up analysis

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Top-down vs bottom-up MS

Top-down – for low complexity samples like single protein or protein complex information at protein level, however less versatile Bottom-up- for omics based studies easy to handle, better separation strategies and lot of automation possible protein inference problem

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Tandem MS (MS/MS) experiment

• MS experiment – Make ions, detect them

• MSMS experiment

– Make ions, do “something” with the ions, detect the products • Peptide/ protein isolation • Peptide/ protein fragmentation

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Things offered in core facility • TOF platform (top-down ) – turn around time within two days

– Total protein mass ,limited proteolysis – Lipids, small molecules and oligos – Native mass spectrometry of intact protein complex

• qExactive HF (bottom-up) – turn around time within 3 weeks – Proteomics , interaction proteomics, phosphoprotoemics, limited

proteolysis – Occasional specialized applications – Cross-link mass spectrometry

• Protein/protein • Protein/RNA

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Protein analysis using gels

The weight of protein cannot be more precise than few 10s of Kda Gel does not provide high precision purity estimate

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Why MS for protein analysis

• Mass resolution and accuracy • 10,000 western blots; antibodies?? • Relative quantification more precise than dye

based methods • Sensitivity – up to low attomoles of peptides • Analysis of PTMs • Translational regulation vs transcriptional

regulation

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Benefits of mass accuracy

• Modern mass spectrometers generally have accuracy to the level of few parts per million – ppm

• Measurement of mass of 100000 Da protein with a mass precision of 10 ppm = 100000 ± 1 Da

Measured Masss at 1 ppm at 10 ppm at 1 ppm

100 kDa 10 Da 1 Da 0.1 Da 10 kDa 1 Da 0.1 Da 0.01 Da 1 kDa 0.1 Da 0.01 Da 0.001 Da

Deviation

Precise mass measurements help in analyzing modifications on the protein resulting in altered protein mass

100 ppm 10 ppm 1 ppm

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Protein ID + purity check in MS

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Liquid chromatography in LC-MS

• Additional dimension of separation prior to MS analysis

• Reversed phase chromatography is ideal to provide optimal ionization of proteins

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LC-MS output file 01

Infrastructure Service Issues Outlook

Chromatogram

Raw spectra

Deconvoluted spectra

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Top- down native MS

Denatured H.pylori urease (alpha 26.6 kDa, orange) and (beta 61.7 kDa, magenta) monomers of urease

Native H.pylori urease 1063.4 kDa

Nature Methods 5, 927 - 933 (2008)

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Native mass spectrometry

• Analysis of intact protein complexes and subunits

• Preserve the native state = no reversed phase chromatography

• Mainly offline analysis • Success depends on the nature of proteins

analyzed

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Native MS for GroEL

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(Shotgun) proteomics workflow

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(Shotgun) proteomics workflow

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(Shotgun) proteomics workflow

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Modes of quantitation

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SILAC vs label free: What to choose

SILAC • Least experimental error

possible • Can mix samples before

extensive processing • Very precise quantification of

less than 2 fold changes • Perfect for protein turnover

projects

• Increased complexity for MS • Labeling step required

Label free • For any sample no labeling

step • Easy for interaction studies • Does not increase the

complexity for MS analysis • Perfect for proteome

quantification, pull down

• Sample preparation should be reproducible

• Not as good as SILAC for < 2 fold change

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Types of projects - omics 1. Expression proteomics 2. PTM analysis at omics level 3. Interaction proteomics 4. PTM analysis for a targeted protein 5. Others – structure, ID verification, purity

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Expression proteomics

• Whole proteome comparison of different systems – Organism, strains, tissues, cells, organelle – verification of knockdowns and off-target/on-target

effects (abundant proteins)

• Very high success rate and easiest to do

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Routine analysis of yeast proteome

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Singleshot HeLa cell proteome

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PTM analysis at omics level • Quantitative changes in PTMs at a

global/large scale – Phospho on S,T,Y (using titanium beads) – Ubiquitination (glygly-motif antibody) – Smt3 (but not mammalian SUMO) – In principle for any PTM that has reliable enrichment strategy, and

detection strategy in mass spectrometer

• Should usually be coupled with quantitation at protein level

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PTM typical workflow

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Site-specific PTM mapping

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PTM analysis – concerns • Sample related

– Sub-stoichiometric - requires enrichment – At least 100 fold more starting material

• Quantitation – Many steps in enrichment step could affect

quantitative accuracy – Single quantitative evidence (Unlike protein that has

many peptides for quantitation)

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PTM analysis – specific protein • Trickier for lower abundant protein in a given sample • Ideally complete coverage is required for the analysis –

sometimes not achievable in practice

• Protein sequence, protease – critical for coverage, and identification of modification sites

• SILAC – depends on protease choice • In need of new methods

– manipulating ions in mass spectrometer – data acquisition strategies

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Interaction proteomics

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Interaction proteomics • Should be straight forward pull down and

analysis by LC-MSMS analysis

• However relatively low success rate in terms of “next step forward” for users with the help of MS results

• Many handling steps = more variation

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IP samples - variety • Magnetic bead based pull downs

• Coupling of antibodies to beads – Bead incompatibility with digestion buffers

• Ni-NTA pull down, flag IP, biotin/avidin IP

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Typical problems in IP • Running a gel for low amount of

sample dilutes the protein • Identify phospho and all other

PTMs • Lack of reproducible pull downs

– Gel as documentation helps in trouble shooting

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Affinity & SILAC - yes or no

• Sample mixing at early stage is possible • The bait/prey is the actual interest in study

– Ubiquitinome– covalent binding– early mixing possible – maximum enrichment required

– Ubiquitin interactome -non-covalent interaction with Ubiquitin – early mixing not possible – minimal enrichment enough

• SILAC for reliable quantitation – 20% change – SILAC is very precise – 20 fold change - SILAC not required

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Enrichment vs Purification

https://www.youtube.com/watch?v=SxjhYzdBEsw

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Limited proteolysis: remaining structure

N terminus C terminus N terminus C terminus

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Summary

• MS facility – top down (same day or within 2 days) and bottom-up offered (within 3 weeks)

• PTM analysis requires enrichment and higher starting material

• IP samples are trickier to handle • Whole proteome quantification is

straightforward

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Thank you for your attention