2. US HUPO Wrap-Up
“my QC is bigger than yours”
~ or ~
The wide world of label-free
quantification
3. Sunday – Lee Hood Keynote
Systems Innovation P4 Challenge Strategies
Diagnostics Wellness Cancer
Holy
Trinity
Transforming
Mechanism Network Exciting Disease Blood
PTSD Technology Protein Medicine Transition
Opportunity Dynamics Genetic Tools Biomarker
4. Monday – Aled Edwards (SGC)
Plenary
• “We are all sheep” – Scientists tend to study the
same small subsets of proteins
• Collaborating with industry to develop chemical
probes (antagonists/inhibitors) for entire class(es)
of enzymes
• Release “open source” through Sigma
5. Monday – Aled Edwards (SGC)
Plenary
• Also working on “high quality” antibodies
– 30% of targets could not be endogenously
IP’d
– Haven’t compared their antibodies with
Uhlen’s
6. Monday – Mechanisms in
Disease
• Mike Tyers (Hospital for Sick Children)
– Cross-linking to detect weak interactions in
Ubiquitin/Proteasome system
– E3/CDK inhibitor binding is modulated by
phosphorylation in disordered regions
– Developed inhibitor of E2 enzyme which
causes tighter binding to Ubiquitin; “Ubiquitin
trap”
7. Monday – Mechanisms in
Disease
• Chris Overall (UBC)
– Studying endogenous “neo-N termini” from
proteolytic cleavage
• 50% of proteins in skin are endogenously cleaved
– Focusing on “rare” cell types/tissues
• Erythrocytes, platelets, placenta, gingiva, B
lymphocytes
– Identified stabilizing N-end acetylation which
helps discern processed proteins from
degradation products
8. Monday – Mechanisms in
Disease
• Mike Snyder (Stanford)
– “Snyderome”: Personal profiling of blood/urine/etc
• 48 months, 73 timepoints, 6 viral infections covered
– Can see dynamics of disease via
transcriptome/microbiome
• “Whole system change” when sick
– “Poop proteome”: ~300 proteins, mostly micro
– TMT labeling of cell lines from 90 patients to look at
protein abundances
• ~5.5% change over time
• Pearson corr. ~0.2 vs. RNA
9. Monday – Emerging Omic
Analysis Technology
• Mike MacCoss (UW)
– Data independent acquisition (DIA) is fast
enough to cover relevant peptide mass range
in less than 3 seconds.
– Adding multiplexing and computational
analysis improves identification, comparable
to DDA.
– DDA is dead. (ok, he didn’t say this out loud).
10. Monday – Emerging Omic
Analysis Technology
• Ben Collins – SWATH 1
– Sam to present
• Christie Hunter – SWATH 2
– Sam to present
11. Monday – Experimental
Quality Control
• Susan Abbatiello (Broad Institute)
– “Variability is additive”
– Developed SRM quality control “system
suitability protocol” based on 15 platforms, 11
labs
– Michrom standards: 6 bovine peptides
– Integrated as plugin to Skyline/Panorama
12. Monday – Experimental
Quality Control
• J. Will Thompson (Duke)
– Measure your variability
– Label-free is most impacted by variability
– Minimize desalting and drying
– Keep mass/volume of lysis, volume & protein ratio of
digest constant across preps
– Include both protein and peptide standards
– LC & MS variability are so low that tech. repl. are
“waste of time”
– Run pools of samples at beginning, middle, and end
with singlets interspersed
– Use PCA to analyze variance. Should show QC runs
in center with biol. variance on outer fringes
13. Monday – Experimental
Quality Control
• Rich Rogers (Amgen)
– Using mass spectrometry to do quality control
of commercial-scale preps
– Accountability
– Detect known and unknown problems in
manufacturing pipeline
14. Monday – Lunch seminar
(Thermo)
• Doing PRM on the Q-exactive
– Use MS2 level for quantification
– Trap light first, then fill with heavy
– Inject both L/H together and analyze in one
scan
– Normalize MS2 intensities based on AGC/fill
time
15. Monday – Bioinformatics
• David Tabb (Vanderbilt) - Quameter
– Four metrics for “rapid QC”: XIC-FWHM-Q2, RT-TIC-Qx, MS1-
Density-Q2, MS2-Freq-Max
– Plot metrics in Principle Component space
• David Shteynberg (ISB) – reSpect
• Sam Payne (PNNL) – Top down
– Informed Quantitation (IQ) for doing label-free quant.
– Match theoretical/actual spectra, then compare charge states
• Vladislav Petyuk (PNNL) – MS/MS IDs in R
– “Data analysis in R is perfectly reproducible”
– MSnID to link identifications to spectral count tools
• Quality filters, convenience functions, FDR
• Ouputs MSnSET object which is understood by other bioconductor
packages
16. Monday – John Yates Plenary
• “I have the biggest protein
list in the room”
• BONCAT pulsed labeling to
look at newly synthesized
proteins
• Pulse label with Methionine
analog, Click with
Biotin, pull down at peptide
level
18. Tuesday – Frontiers in
Instrumentation
• Vlad Zubrouskov (Thermo)
– Fusion is the fastest CID ion trap instrument
ever built.
– “Pipeline” means Full Orbi-MS1 scans at
same time as CID IT-MS2 scans.
– More yeast peptides identified than ever
before in a single run.
– DDA is not dead. (He really said this.)
19. Tuesday – Statistical Analysis
Proteomics
• Olga Vitek (Purdue) – MSstats
– R package for statistical relative quantification
– Generalizes ratio-based models, robust to
missing values
– Supports varied experimental
designs/methods
– Integrated into Skyline as plugin
20. Tuesday – Statistical Analysis
Proteomics
• Steven Skates (Mass. Gen.) – Models
– Developing statistical models specifically for
mass spec biomarker discovery
– BUGS (Bayesian
inference Using Gibbs Sampling), JAGS (Just
Another Gibbs Sampler), STAN (probabilistic
programming language)
21. Tuesday – Statistical Analysis
Proteomics
• Attila Kertesz-Farkas (UW) – Cascade
Search
– Nested databases
– See ~10% improvement in PSMs
Fully
tryptic
Semi
tryptic
Non-
enzymatic
22. Tuesday – Protein Interactions
and Structure
• Michael Washburn (Stowers Inst.) – IP
– Correct for background
• Ileana Cristea (Princeton) – Viral Infection
– Identified viral DNA sensor in nucleus
– Virus encodes E3 ubiquitin ligase targeting
sensor protein
• Jim Bruce (UW) – Protein interaction
reporters
Tag-1 Target
Tag-1 Tag-2 Target
23. Tuesday – Aging Proteomics
• Brad Gibson (Buck Inst.) – Acetylation targets of
SIRT3
– NAD+-dependent deacetylase
– Many core enzymes are acetylated by SIRT5
• Peter Rabinovich (UW) – Rapamycin as mimic of
caloric restriction
• Sina G. – Turnover kinetics
– Protein half lives: Brain ~ 9 days, Liver ~ 3 days
– No dramatic change in turnover between old/young mice
– Subset of mitochondrial proteins turns over quickly in old
mice
– Drosophila show decreased global turnover in old flies
– Activating autophagy in flies increases lifespan
24. Tuesday evening workshop
• Big Data – Big Hang-ups?
– More discussion needed in the community to
inform and encourage participation.
– Chorus as an alternative to Tranche.
• But is riddled with Nathan Yates gluFib runs.
– Metadata annotations
• Not immediately necessary.
• Community will determine the most important
metadata.
• Big Data can still be used without annotations, i.e. stop
looking at everything as individual experiments with
specific context.
25. Wednesday – Focus on Global
Health
• Stefan Kappe (SBRI) – Malaria
– Antibody against CSP is in phase III, ~40% effective
– Identified ~1800 proteins in 3 types of sporozoite
– Identified several surface proteins
– SSP3: surface protein accessible after shedding of CSP
• Olga Schubert (ETH Zurich) – MTB proteomics
– Extensive shotgun/fractionation covered 77% of proteome
– Synthetic peptides/SRM increased to 97% of proteome
– Generated high-quality spectral library
– 67% of proteome detected in single SWATH run
• Brandy Young (Univ. of Cape Town) – TB
Biomarkers
26. New Investigator Award –
Judit Villen
• Developing “wiring diagrams” of
phosphorylation pathways
• Developing new tools to assign phospho.
sites
– Manually assigned 50k spectra in grad school
(so stop bitching and get to work).
27. Wednesday Keynote – Hudson
Freeze
• Use MALDI signature of reporter protein
transferrin to detect glycosylation defects
in patients
• Follow up with whole-exome sequencing
• Develop cell lines from patients to do
functional assays
• Can treat a few with dietary supplement
