One Shot Proteomics
Prof. Peter B. O’Connor, Dr. Maria van Agthoven, Dr. Mark Barrow, Federico Floris, Haley Simon
Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK
Proteomics often runs into two problems: 1) precursor ions selection windows can admit more than one precursor ion for fragmentation,
resulting in confusion of which fragment comes from which precursor and 2) ion selection and MS/MS are serial processes requiring a decision
about which ions are important to fragment. A new tandem mass spectrometry method, which addresses these problems, called 2-dimensional
(2D) mass spectrometry, is now possible as shown below. Here we present the first proteomics data using 2DMS methods.
Collagen sequence (409 kDa)
α1-[1] = 138, 938 Da
MFSFVDLRLL
DVWKPVPCQI
SPTDQETTGV
GPPGLGGNFA
QGPPGEPGEP
ARGLPGTAGL
GPRGLPGERG
KGEGGPQGPR
APGIAGAPGF
GPTGIQGPPG
AGPKGPAGER
KTGPPGPAGQ
GPPGAVGPAG
PGEAGKPGEQ
APGNDGAKGD
GADGAPGKDG
RGEPGPPGPA
PIGNVGAPGP
GKEGSKGPRG
QGIAGQRGVV
PPGLAGPPGE
GAPGPVGPAG
QGDRGIKGHR
KDGLNGLPGP
PQPPQEKAHD
RKNPARTCRD
PTQPSVAQKN
LTFLRLMSTE
GNSRFTYSVT
QEFGFDVGPA
LLLAATALLT
CVCDNGNVLC
EGPKGDTGPR
PQLSYGYDEK
GASGPMGPRG
PGMKGHRGFS
RPGAPGPAGA
GSEGPQGVRG
PGARGPSGPQ
PAGEEGKRGA
GAPGPAGPKG
DGRPGPPGPP
KDGEAGAQGP
GVPGDLGAPG
AGAPGAPGSQ
VRGLTGPIGP
GFAGPPGADG
KGARGSAGPP
ETGPAGRPGE
GLPGQRGERG
SGREGAPGAE
KSGDRGETGP
GFSGLQGPPG
IGPPGPRGRT
GGRYYRADDA
LKMCHSDWKS
WYISKNPKEK
ASQNITYHCK
YDGCTSHTGA
CFL
HGQEEGQEEG
DDVICDELKD
GPRGPAGPPG
STGISVPGPM
PPGPPGKNGD
GLDGAKGDAG
RGNDGATGAA
EPGPPGPAGA
GPSGPPGPKG
RGEPGPAGLP
SPGEAGRPGE
GARGQAGVMG
PGPAGPAGER
PSGARGERGF
GAPGLQGMPG
PGPAGAPGDK
QPGAKGEPGD
GATGFPGAAG
VGPPGPPGPA
FPGLPGPSGE
GSPGRDGSPG
AGPAGPIGPV
PPGSPGEQGP
GDAGPAGPPG
NVVRDRDLEV
GEYWIDPNQG
RHVWYGESMT
NSVAYMDQQT
WGKTVIEYKT
QEEDIPPVTC
CPNAKVPTDE
RDGIPGQPGL
GPSGPRGLPG
DGEAGKPGRP
PAGPKGEPGS
GPPGPTGPAG
AGPAGNPGAD
NSGEPGAPGS
GPPGERGGPG
AGLPGAKGLT
FPGPKGAAGE
GEQGPAGSPG
PGERGVQGPP
ERGAAGLPGP
GEAGPSGPAG
AGAKGDAGPP
RVGPPGPSGN
GEKGAPGADG
PGKQGPSGAS
AKGDRGETGP
GARGPAGPQG
SGASGPAGPR
PPGPPGPPGP
DTTLKSLSQQ
CNLDAIKVFC
GGFQFEYGGQ
GNLKKALLLQ
TKTSRLPIID
Collagen (409 kDa) 1D MS
Collagen (409 kDa) 1D MS/MS
α2-[1] = 129, 064 Da
VQNGLRYHDR
CCPVCPEGQE
PGPPGPPGPP
PPGAPGPQGF
GERGPPGPQG
PGENGAPGQM
PPGFPGAVGA
GQPGAKGANG
KGDTGAKGEP
SRGFPGADGV
GSPGSPGPDG
PGKAGERGVP
FQGLPGPAGP
GPAGPRGANG
KGDRGDAGPK
PTGARGAPGD
GPAGPAGPPG
AGPPGPPGPA
PAGAPGTPGP
GERGPPGPMG
AGPPGAPGAP
PRGDKGETGE
GPPGSAGSPG
PSGGYDLSFL
IENIRSPEGS
NMETGETCVY
GSDPADVAIQ
GSNEIEIRAE
VAPLDVGAPD
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1463
MLSFVDTRTL
GRDGDDGIPG
GASGAPGPQG
PGERGVVGPQ
APGENGTPGQ
GPPGFPGAPG
NGLPGAKGAA
SKGESGNKGE
GSRGLPGADG
PGSPGNIGPA
DPGKAGEKGH
GFQGLPGPAG
TGPIGSRGPS
GKGEKGETGL
GPAGPRGSPG
VGPTGPVGAA
ISGPPGPPGP
GTAGPPGTPG
PGARGPPGNV
NAGPVGAAGA
GIRGDKGEPG
RGPAGPSGPA
PSGGGYEFGF
EGSRKNPART
CIRAQPEDIP
QLAFMRLLAN
EGNSRFTYTV
DQEIRLNIGP
LLLAVTSCLA
PPGPPGPPGP
FQGPPGEPGE
GARGFPGTPG
TGARGLPGER
PKGELGPVGN
GLPGVAGAPG
PGAVGQPGPP
RAGVMGPAGS
GKEGPVGLPG
AGLAGARGAP
TAGEAGKPGE
GPPGPDGNKG
RGDIGSPGRD
ERGEVGPAGP
GPSGPNGPPG
AGKEGLRGPR
PQGLLGAPGF
GNPGVNGAPG
PGPQGPVGPV
DKGPRGLPGL
GKDGRIGQPG
DGDFYRADQP
CRDLRLSHPE
VKNWYRNSKA
HASQNITYHC
LVDGCSKKTN
VCFK
TCQSLQEATA
PGLGGNFAAQ
PGQTGPAGAR
LPGFKGIRGH
GRVGAPGPAG
PGPAGPAGPR
LPGPRGIPGP
GPSGEEGKRG
RGATGPAGVR
IDGRPGPIGP
GPDGNNGAQG
RGIPGEFGLP
EPGVVGAPGT
GARGAPGAIG
NGFAGPAGAA
PAGSRGDGGP
GDQGPVGRSG
LGLPGSRGER
EAGRDGNPGN
GKHGNRGEPG
KGHNGLQGLP
AVGPAGIRGS
RSPTSLRPKD
WSSGYYWIDP
KKHVWVGETI
KNSIAYMDEE
EWQKTIIEYK
RKGPSGDRGP
FDAKGGGPGP
GPPGPPGKAG
NGLDGLKGQP
ARGSDGSVGP
GEVGLPGLSG
VGAAGATGAR
STGEIGPAGP
GPNGDSGRPG
AGARGEPGNI
PPGLQGVQGG
GPAGARGERG
AGPSGPSGLP
APGPAGANGD
GQPGAKGERG
PGATGFPGAA
ETGASGPPGF
GLPGVAGSVG
DGPPGRDGQP
PAGAVGPAGA
GLAGHHGDQG
QGSQGPAGPP
YEVDATLKSL
NQGCTMDAIK
NGGTQFEYNV
TGNLKKAVIL
TNKPSRLPIL
RGERGPPGPP
MGLMGPRGPP
EDGHPGKPGR
GAPGVKGEPG
VGPAGPIGSA
PVGPPGNPGA
GLVGEPGPAG
PGPPGLRGNP
EPGLMGPRGF
GFPGPKGPSG
KGEQGPAGPP
PPGESGAAGP
GERGAAGIPG
RGEAGPAGPA
TKGPKGENGP
GRTGPPGPSG
VGEKGPSGEP
EPGPLGIAGP
GHKGERGYPG
VGPRGPSGPQ
APGAVGPAGP
GPPGPPGPPG
NNQIETLLTP
VYCDFSTGET
EGVTTKEMAT
QGSNDVELVA
DIAPLDIGGA
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1364
Pink – Appears in MS
Underlined – Sequenced by MS/MS
Blue – Telopeptide region
Shaded – Cross-linking region
2DMS Scan Lines
The principle of 2DMS
2DMS of Collagen (409 kDa)
Ion cyclotron radius modulation by introducing a delay
between two identical pulses:
rA = r0 2(1 + cos(ωiont1 )
De-excitation
pulse (P2= P1)
Excitation
pulse (P1)
Incremental
delay
(t1)
Pre-detection
pulse (P3)
(IRMPD, ECD)
(τm)
No
Fragments
FT (t1)
Amplitude (a.u.)
∆ϕ = 2n ×180°
∆ϕ = (2n + 1) ×180°
Fragments
0
Amplitude (a.u.)
IRMPD
FT (t1)
Detection
(t2)
500
1000
Comparison of 2DMS and 1DMS
1500
precursor ICR frequency
100
ICR
cell
• NO isolation
• YES fragmentation
Precursor ion
own ICR frequency
100
80
60
40
20
0
• All ions in a complex
sample fragmented and
visualised on one
spectrum
Cleavage coverage (%)
∆ϕ = ωion × t1 + ϕ0
80
60
40
20
0
0
500
1000
Frequency (kHz)
1500
80
70
60
50
40
30
20
10
0
CAD
ECD
IRMPD
2D-MS
Technique
Fragment ion
Fourier transform according to t1 (delay between P1 and P2) and t2
(measurement date):
MS/MS precursor and fragment ion spectra of all ion species
2DMS of Collagen (409 kDa)
Precursor m/z 951.8092 | [α1 (763-795) + 3H] 3+ | 33 A.A. length
2DMS of Calmodulin (17 kDa) Tryptic Digest
2DMS of Yeast tryptic digest.
Raw IRPMD
spectrum, no
denoising.
Total acquisition
time ~40 minutes.
Top-Down 2DMS of Calmodulin (17 kDa)
Acknowledgements
Conclusions
• 2-Dimensional mass spectrometry of proteomic samples is
possible. The 2DMS method generates fragment ions for all ions
at the same time, so it avoids the ion preselection bias and
multiple ion selection problems of traditional MS/MS methods.
• There are a lot of issues to solve, including stabilizing the method,
calibrating the spectra, picking peaks and interpreting spectra, but
the method works.
• Thus, we can now get an entire proteome in ‘one shot’ with a
2DMS experiment.
• ‘One Shot’ proteomics is possible.
People
Dr. Huilin Li, Dr. Yulin Qi, Dr. Andrea LopezClavijo, Dr. Pilar Perez-Hurtado, Meng Li,
Samantha Benson, Dr. Rebecca Wills, Dr.
Mark Barrow, Dr. Ron Heeren, Matthew
Griffiths, Dr. David Kilgour, Dr. Terry Lin,
Andrew Soulby, Dr. Juan Wei, Chris
Wootton, Hayley Simon, Dr. Maria van
Agthoven, Cookson Chiu, Federico Floris,
Dr. Parminder Kaur, Dr. Raman Mathur, Dr.
Jason Cournoyer, Dr. Nadia Sargaeva, Dr.
Xiaojuan Li, Dr. MD. Jason Pittman, Dr.
Bogdan Budnik, Dr. Susanne Moyer, Dr.
Vera Ivelva, Dr. Konstantine Aizikov,
Haytham Hussein, Mark Kozlowski, Samuel
Peel, Yuko (Pui Yiu) Lam, Holly Chan, Man
Ying Wong.
• Warwick Postgraduate
Research Scholarship
(WPRS)
• Warwick Centre for
Analytical Science
• Protein Biology and
BioPhysics Network
• Bruker
• NIH
• ERC
• EPSRC
2DMS of Yeast tryptic digest, expansion of the ~600-700 Da
(precursor ion) and ~750-950 Da (product ion) region.