Transcript Faster, More Sensitive Peptide ID by Sequence DB Compression

Proteomics and
Glycoproteomics
(Bio-)Informatics
of Protein Isoforms
Nathan Edwards
Department of Biochemistry and
Molecular & Cellular Biology
Georgetown University Medical Center
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
2
Mass Spectrometer
Sample
+
_
Ionizer
• MALDI
• Electro-Spray
Ionization (ESI)
Mass Analyzer
• Time-Of-Flight (TOF)
• Quadrapole
• Ion-Trap
Detector
• Electron
Multiplier
(EM)
3
Mass Spectrum
4
Mass is fundamental
5
Sample Preparation for MS/MS
Enzymatic Digest
and
Fractionation
6
Single Stage MS
MS
7
Tandem Mass Spectrometry
(MS/MS)
Precursor selection
8
Tandem Mass Spectrometry
(MS/MS)
Precursor selection +
collision induced dissociation
(CID)
MS/MS
9
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
10
Unannotated Splice Isoform

Human Jurkat leukemia cell-line



LIME1 gene:


LCK interacting transmembrane adaptor 1
LCK gene:



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Lipid-raft extraction protocol, targeting T cells
von Haller, et al. MCP 2003.
Leukocyte-specific protein tyrosine kinase
Proto-oncogene
Chromosomal aberration involving LCK in leukemias.
Multiple significant peptide identifications
11
Unannotated Splice Isoform
12
Unannotated Splice Isoform
13
Translation start-site correction

Halobacterium sp. NRC-1



GdhA1 gene:

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Extreme halophilic Archaeon, insoluble membrane
and soluble cytoplasmic proteins
Goo, et al. MCP 2003.
Glutamate dehydrogenase A1
Multiple significant peptide identifications

Observed start is consistent with Glimmer 3.0
prediction(s)
17
Halobacterium sp. NRC-1
ORF: GdhA1


K-score E-value vs PepArML @ 10% FDR
Many peptides inconsistent with annotated
translation start site of NP_279651
0
40
80 120 160 200 240 280 320 360 400 440
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What if there is no "smoking
gun" peptide…
20
What if there is no "smoking
gun" peptide…
21
What if there is no "smoking
gun" peptide…
22
HER2/Neu Mouse Model of
Breast Cancer


Paulovich, et al. JPR, 2007
Study of normal and tumor mammary tissue
by LC-MS/MS


Peptide-spectrum assignments
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

Normal samples (Nn): 161,286 (49.7%)
Tumor samples (Nt): 163,068 (50.3%)
4270 proteins identified in total

23
1.4 million MS/MS spectra
2-unique generalized protein parsimony
Nascent polypeptide-associated
complex subunit alpha
7.3 x 10-8
24
Pyruvate kinase isozymes M1/M2
2.5 x 10-5
25
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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[195.00-2000.00]
MS yr_inclusion
60
40
20
CID Protein Fragmentation
Spectrum from Y. rohdei
21.03 21.46
0
19.5
20.0
20.5
21.0
21.5
22.0
22.5
Time (min)
yr_inclusion #1937-2437 RT: 19.45-24.36 AV: 21 NL: 4.80E4
F: FTMS + p ESI d Full ms2 [email protected] [195.00-2000.00]
576.83
z=2
100
23.0
23.5
24.0
24.5
25.0
756.70 +8 MW 6044.11
90
80
70
584.57
z=4 720.39
z=2
60
50
785.41
z=4
40
694.62
z=4
30
20
10
840.16
z=7
200.78 329.71
z=?
z=?
903.81
z=3
928.49
z=4
461.16 559.55
z=4
z=?
992.53
z=3
555.29
z=4
0
200
400
600
800
1118.93
z=?
1000
1253.14
z=? 1345.30
z=?
1200
1400
1804.48
z=?
1491.23 1610.27 1666.89
1883.75
z=?
z=?
z=?
z=?
1600
1800
2000
m/z
27
60
CID Protein Fragmentation
Spectrum from Y. rohdei
[email protected]
[195.00-2000.00]
MS yr_inclusion
40
20
21.03 21.46
0
19.5
20.0
20.5
21.0
21.5
22.0
22.5
Time (min)
yr_inclusion #1937-2437 RT: 19.45-24.36 AV: 21 NL: 4.80E4
F: FTMS + p ESI d Full ms2 [email protected] [195.00-2000.00]
576.83
z=2
100
23.0
23.5
24.0
24.5
25.0
756.70 +8 MW 6044.11
90
Match to Y. pestis 50S
Ribosomal Protein L32
80
70
584.57
z=4 720.39
z=2
60
50
785.41
z=4
40
694.62
z=4
30
20
10
840.16
z=7
200.78 329.71
z=?
z=?
903.81
z=3
928.49
z=4
461.16 559.55
z=4
z=?
992.53
z=3
555.29
z=4
0
200
400
600
800
1118.93
z=?
1000
1253.14
z=? 1345.30
z=?
1200
1400
1804.48
z=?
1491.23 1610.27 1666.89
1883.75
z=?
z=?
z=?
z=?
1600
1800
2000
m/z
28
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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Identified E. herbicola proteins

DNA-binding protein HU-alpha
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m/z 732.71, z 13+, E-value 7.5e-26, Δ -14.128
Eight proteins identified with "large" |Δ|
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Identified E. herbicola proteins
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DNA-binding protein HU-alpha
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m/z 732.71, z 13+, E-value 7.5e-26, Δ -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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Phylogenetic placement of
E. herbicola
Phylogram
Cladogram
phylogeny.fr – "One-Click"
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Glycoprotein
Microheterogeneity
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Glycosylation is important, but our analytic
tools are rather rudimentary
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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…
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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
Fut-VI
(α1-3 Fuc)
Higai,2008
Fut-VIII
NH3+
(α1-6 Fuc)
Comunale,
2010
N
X
S/T
ST-VI Gal1
(α 2-6 NeuAc)
Hedlund, 2008
GnT-V
(β1-6 GlcNAc)
Wang, 2007
COOK. Chandler
The informatics challenge

Identify glycopeptides in large-scale tandem
mass-spectrometry datasets
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Good, but not great, instrumentation
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Many glycopeptide enriched fractions
Many tandem mass-spectra / fraction
QStar Elite – CID, good MS1/MS2 resolution
Strive for hypothesis-generating analysis
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Site-specific glycopeptide characterization
Glycoform occupancy in differentiated samples
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CID Glycopeptide Spectrum
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Observations
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Oxonium ions (204, 366) help distinguish
glycopeptides from peptides…
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Few peptide b/y-ions to identify peptides…
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…but do little to identify the glycopeptide
…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 (HPT_HUMAN)
Haptoglobin Standard
MVSHHNLTTGATLINE
NLFLNHSE*NATAK
VVLHPNYSQVDIGLIK
•
*
N-glycosylation motif (NX/ST)
Site of GluC cleavage
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Pompach et al. Journal of Proteome Research 11.3 (2012): 1728–1740.
Tuning the filters…

We estimate the number of false-positives…
…so that the user can tune the search parameters
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Application of Exoglycosidases
to locate Fucose
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At ITIH4 site N517
LPTQNITFQTE
LPTQNITFQTE
LPTQNITFQTE
LPTQNITFQTE
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K. Chandler
NVVFVIDK ITIH4 Glycopeptide
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K. Chandler
Similar Glycopeptides Spectra
( mass Δ ~ +162 Da)
?
+162 Da
MVSHHNLTTGATLINE
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Fragmented Glycopeptides
( mass Δ ~ +162 Da)
?
MVSHHNLTTGATLINE
+162 Da
MVSHHNLTTGATLINE
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Propagating Annotations
•MVS+A2G2
•MVS+A1G1
•MVS+A2G2
•VVL+A1G1
•MVS+A2G2
•VVL+A2G2
•MVS+A1G1
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G. Berry
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
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NSF Graduate
Fellowship (Chandler)
Funding: NCI
Fenselau lab (UMD)



Kevin Chandler
Gwenn Berry

Colin Wynne
Avantika Dhabaria
Goldman lab (GU)
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
Kevin Chandler
Petr Pompach
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