Proteomics and Glycoproteomics (Bio-)Informatics of Protein Isoforms

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Proteomics and Glycoproteomics(Bio-)Informatics
of Protein Isoforms

• Nathan Edwards
• Department of Biochemistry and Molecular
  Cellular Biology
• Georgetown University Medical Center

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Outline

• Tandem mass-spectrometry of peptides
• Detection of alternative splicing protein
  isoforms
• Phyloproteomics using top-down mass-spec.
• Characterization of glycoprotein
  microheterogeneity by mass-spectrometry

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Mass Spectrometer

• ElectronMultiplier(EM)

• Time-Of-Flight (TOF)
• Quadrapole
• Ion-Trap

• MALDI
• Electro-SprayIonization (ESI)

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Mass Spectrum
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Mass is fundamental
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Sample Preparation for MS/MS
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Single Stage MS
MS
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Tandem Mass Spectrometry(MS/MS)
Precursor selection
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Tandem Mass Spectrometry(MS/MS)
Precursor selection collision induced
dissociation (CID)
MS/MS
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Why Tandem Mass Spectrometry?

• MS/MS spectra provide evidence for the amino-acid
  sequence of functional proteins.
• Key concepts
• Spectrum acquisition is unbiased
• Direct observation of amino-acid sequence
• Sensitive to small sequence variations

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Unannotated Splice Isoform

• Human Jurkat leukemia cell-line
• Lipid-raft extraction protocol, targeting T cells
• von Haller, et al. MCP 2003.
• LIME1 gene
• LCK interacting transmembrane adaptor 1
• LCK gene
• Leukocyte-specific protein tyrosine kinase
• Proto-oncogene
• Chromosomal aberration involving LCK in
  leukemias.
• Multiple significant peptide identifications

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Unannotated Splice Isoform
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Unannotated Splice Isoform
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Splice Isoform Anomaly

• Human erythroleukemia K562 cell-line
• Depth of coverage study
• Resing et al. Anal. Chem. 2004.
• Peptide Atlas A8_IP
• SALT1A2 gene
• Sulfotransferase family, cytosolic, 1A
• 2 ESTs, 1 mRNA
• mRNA from lung, small cell-cancinoma sample
• Single (significant) peptide identification
• Five agreeing search engines
• PepArML FDR lt 1.
• All source engines have non-significant E-values

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Splice Isoform Anomaly
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Splice Isoform Anomaly
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Translation start-site correction

• Halobacterium sp. NRC-1
• Extreme halophilic Archaeon, insoluble membrane
  and soluble cytoplasmic proteins
• Goo, et al. MCP 2003.
• GdhA1 gene
• Glutamate dehydrogenase A1
• Multiple significant peptide identifications
• Observed start is consistent with Glimmer 3.0
  prediction(s)

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Halobacterium sp. NRC-1ORF GdhA1

• K-score E-value vs PepArML _at_ 10 FDR
• Many peptides inconsistent with annotated
  translation start site of NP_279651

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Translation start-site correction
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What if there is no "smoking gun" peptide
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What if there is no "smoking gun" peptide
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What if there is no "smoking gun" peptide
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HER2/Neu Mouse Model of Breast Cancer

• Paulovich, et al. JPR, 2007
• Study of normal and tumor mammary tissue by
  LC-MS/MS
• 1.4 million MS/MS spectra
• Peptide-spectrum assignments
• Normal samples (Nn) 161,286 (49.7)
• Tumor samples (Nt) 163,068 (50.3)
• 4270 proteins identified in total
• 2-unique generalized protein parsimony

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Nascent polypeptide-associated complex subunit
alpha
7.3 x 10-8
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Pyruvate kinase isozymes M1/M2
2.5 x 10-5
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Phyloproteomics

• Fragment intact proteins (top-down MS)
• Match the spectra to protein sequences
• Place the organism phylogenetically
• Works even for unknown microorganisms without any
  available sequences

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CID Protein Fragmentation Spectrum from Y. rohdei
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CID Protein Fragmentation Spectrum from Y. rohdei
Match to Y. pestis 50S Ribosomal Protein L32
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Exact match sequence
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Phylogeny Protein vs DNA
Protein Sequence
16S-rRNA Sequence
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What about mixtures?
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Shared Small Ribosomal Proteins
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Shared Small Ribosomal Proteins
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Identified E. herbicola proteins

• DNA-binding protein HU-alpha
• m/z 732.71, z 13, E-value 7.5e-26, ? -14.128
• Eight proteins identified with "large" ?

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Identified E. herbicola proteins

• DNA-binding protein HU-alpha
• m/z 732.71, z 13, E-value 1.91e-58
• Use "Sequence Gazer" to find mass shift
• ?M mode can "tolerate" one shift for free!

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Identified E. herbicola proteins

• DNA-binding protein HU-alpha
• m/z 732.71, z 13, E-value 7.5e-26, ? -14.128
• Extract N- and C-terminus sequence supported by
  at least 3 b- or y-ions

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E. herbicola protein sequences
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E. herbicola sequences found in other species
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Phylogenetic placement of E. herbicola
Cladogram
Phylogram
phylogeny.fr "One-Click"
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Glycoprotein Microheterogeneity

• Glycosylation is important, but our analytic
  tools are rather rudimentary
• Detach glycans (PNGase-F) and analyze glycans
• Detach glycans (PNGase-F) and analyze peptides
• Get glycan structures, but no association with
  protein or protein site, or
• Get glycosylation sites, but no association with
  glycan structures.
• We analyze glycopeptides directly
• Challenges all facets of glycoproteomics

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Altered N-Glycosylation in Cancer
Glycosyltransferase Expression or Glycan Analyses
GalNAc Sialic Acid Gal
GlcNAc Man
K. Chandler
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The informatics challenge

• Identify glycopeptides in large-scale tandem
  mass-spectrometry datasets
• Many glycopeptide enriched fractions
• Many tandem mass-spectra / fraction
• Good, but not great, instrumentation
• QStar Elite CID, good MS1/MS2 resolution
• Strive for hypothesis-generating analysis
• Site-specific glycopeptide characterization
• Glycoform occupancy in differentiated samples

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CID Glycopeptide Spectrum
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Observations

• Oxonium ions (204, 366) help distinguish
  glycopeptides from peptides
• but do little to identify the glycopeptide
• Few peptide b/y-ions to identify peptides
• but intact peptide fragments are common
• If the peptide can be guessed, then
• the glycan's mass can be determined

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Haptoglobin Standard

• N-glycosylation motif (NX/ST)
• Site of GluC cleavage

Pompach et al. Journal of Proteome Research 11.3
(2012) 17281740.
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Tuning the filters

• Oxonium ions
• Number intensity
• Match tolerance
• "Intact-peptide" fragments
• Number intensity
• Match tolerance
• Glycan composition
• ICScore
• Constrain search space
• Match tolerance

• Glycan database
• Constrain search space
• Match tolerance
• Precursor ion
• Non-monoisotopic selection
• Sodium adducts
• Charge state
• Peptide search space
• Semi-specific peptides
• Non-specific peptides
• Peptide MW range
• Variable modifications

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Tuning the filters

• We estimate the number of false-positivesso
  that the user can tune the search parameters

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Application of Exoglycosidasesto locate Fucose

• At ITIH4 site N517

K. Chandler
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NVVFVIDK ITIH4 Glycopeptide
K. Chandler
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Similar Glycopeptides Spectra( mass ? 162 Da)
?
162 Da
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Fragmented Glycopeptides( mass ? 162 Da)
?
162 Da
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Propagating Annotations
G. Berry
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Summary

• Mass-spectrometry coupled with protein chemistry
  and good informatics can look beyond the obvious
  to the unexpected...
• and there is plenty to find!

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Acknowledgements

• Edwards lab
• Kevin Chandler
• Gwenn Berry
• Fenselau lab (UMD)
• Colin Wynne
• Avantika Dhabaria
• Goldman lab (GU)
• Kevin Chandler
• Petr Pompach

• NSF Graduate Fellowship (Chandler)
• Funding NCI