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Welcome!

Mass Spectrometry meets CheminformaticsWCMC Metabolomics Course 2014

Tobias Kind

Course: Search of MS/MS files with theNIST MS Search GUI

Biology

Chemistry

Informatics

http://fiehnlab.ucdavis.edu/staff/kindCC-BY License

FT-ICR-MS

Iontrap

Triple-Quad

Q-TOF

Orbitrap

TOF-TOF

low resolution

high resolution

MS/MS spectrum LipidBlast

200k tandem mass spectra

Results withannotation

1) Precursormatch

2) Product ionsearch

Instrumentation Data processing Lipid identificationLipidBlast DB

Report withwith scores

General workflow for MS/MS search

1) The precursor filter removes 99% of all false candidates within a given mass window.2) The product ion match uses traditional similarity scoring of remaining candidates.

NIST compatible libraries are simply copied as subdirectoriesunder the MSSEARCH directory

NIST MS Search GUI (2.0f 2010) – Search parameters

NIST MSPepSearch mass spectral library search program Thanks to Dmitrii V. Tchekhovskoi and Stephen E. SteinMass Spectrometry Data Center; National Institute of Standards and Technology (NIST)Visit http://peptide.nist.gov or http://chemdata.nist.gov for more information,software, or MS libraries.

General Library Search view of NIST MS Search GUI

The NIST11 full version has MS/MS tree view

The NIST MS/MS tree view offers the compound viewwith different ionization voltages

MS/MS search= Precursor filter + similarity filter

precursor ionnot visible

Similarity match of MS/MS spectrum ‐ head to tail view

Head to tail – different spectra 

Side by side view of MS/MS spectrum 

Subtraction view of MS/MS spectrum what are the largest peak differences?

Transfer (right click in NIST MS Search) to MS Interpreter for further inspection

Simple name search – not very powerful

Sequential search – can find anything 

177 spectra with name fragment “galactosyl”

177 spectra with name fragment “galactosyl” ANDpeak at m/z 264 must be between 80% and 100% of base peak

Powerful sequential search

Powerful sequential search: Ten result spectra with modified query 

Formula search – how many formulae in database? 

Num Platforms Num Platforms1 ABI 4000 Q‐Trap 24 JEOL JMS‐HX110A/110A Tandem MS FAB2 ABI 4700 MALDI TOF/TOF 25 Kratos MALDI‐TOF AXIMA‐CFR3 ABI 4800 MALDI‐TOF/TOF 26 SHIMADZU KRATOS MALDI TRAP TOF4 ABI API 2000 triple quadrupole 27 SHIMADZU LCMS‐IT‐TOF5 ABI QSTAR‐XL QTOF 28 Thermo Finnigan DecaXP ion trap6 ABI QTRAP 4000 29 Thermo Finnigan LCQ DECA ion trap7 ABI Sciex API III QQQ 30 Thermo Finnigan LTQ linear ion trap8 ABI‐QSTAR‐Pulsar‐Quadrupol‐TOF 31 Thermo Finnigan TSQ Quantum Triple Quadrupole9 ABI‐QSTAR‐XL‐Quadrupol‐TOF 32 Thermo Fisher Exactive Orbitrap10 Agilent 6410 triple quadrupole MS 33 Thermo Fisher LTQ‐FT11 Agilent 6520 Q‐TOF 34 Thermo LTQ Orbitrap12 Agilent 6530 QqTOF 35 Thermo LXQ iontrap13 Agilent Ion Trap SL 36 Thermo Orbitrap Velos ESI 14 Agilent Ion Trap XCT 37 Waters AutoSpec magnetic sector MS15 Agilent LC/MSD 1100 Ion Trap  38 Waters micro QTOF 16 Agilent MSD 1100 single quadrupole MS 39 Waters Micromass Q‐TOF Micro 17 Agilent QTOF 40 Waters MicroMass QqQ triple quadrupole18 Bruker DESI FTICR APEX‐Q 41 Waters QqQ triple quadropole VG Quattro II19 Bruker Esquire 3000 ion trap 42 Waters QTOF Premier20 Bruker Esquire ion trap 43 Waters Synapt HDMS21 Bruker microTOF qQ‐TOF22 Bruker UltraFlex II MALDI TOF‐TOF23 Bruker ultrafleXtreme MALDI TOF/TOF

LipidBlast platform compatibility

Number positive MS/MS spectra 78314Number negative MS/MS spectra 134202Total MS/MS spectra 212516Total lipid compound number 119200

Number MS/MS spectra Number Number LipidClass Short Number compounds with different adducts MS/MS LIBS No neg MS/MS No pos MS/MS

1 Phosphatidylcholines PC 5476 10952 2 0 109522 Lysophosphatidylcholines lysoPC 80 160 2 0 1603 Plasmenylphosphatidylcholines plasmenyl‐PC 222 444 2 0 4444 Phosphatidylethanolamines PE 5476 16428 3 5476 109525 Lysophosphatidylethanolamines lysoPE 80 240 3 80 1606 Plasmenylphosphatidylethanolamines plasmenyl‐PE 222 666 3 222 4447 Phosphatidylserines PS 5123 15369 3 5123 102468 Sphingomyelines SM 168 336 2 0 3369 Phosphatidic acids PA 5476 16428 3 5476 1095210 Phosphatidylinositols PI 5476 5476 1 5476 011 Phosphatidylglycerols PG 5476 5476 1 5476 012 Cardiolipins CL 25426 50852 2 50852 013 Ceramide‐1‐phosphates CerP 168 336 2 168 16814 Sulfatides ST 168 168 1 168 015 Gangliosides [glycan]‐Cer 880 880 1 880 016 Monoacylglycerols MG 74 148 2 0 14817 Diacylglycerols DG 1764 3528 2 0 352818 Triacylglycerols TG 2640 7920 3 0 792019 Monogalactosyldiacylglycerols MGDG 5476 21904 4 5476 1642820 Digalactosyldiacylglycerols DGDG 5476 10952 2 5476 547621 Sulfoquinovosyldiacylglycerols SQDG 5476 5476 1 5476 022 Diacylated phosphatidylinositol monomannoside Ac2PIM1 144 144 1 144 023 Diacylated phosphatidylinositol dimannoside Ac2PIM2 144 144 1 144 024 Triacylated phosphatidylinositol dimannoside Ac3PIM2 1728 1728 1 1728 025 Tetraacylated phosphatidylinositol dimannoside Ac4PIM2 20736 20736 1 20736 026 Diphosphorylated hexaacyl Lipid A LipidA‐PP 15625 15625 1 15625 0

Total All libraries 119200 212516 50 134202 78314

LipidBlast compound coverage

Example Thermo LTQ; PC 34:1; [M+H]+ ; PC 34:1

Name: Agilent Ion Trap XCT ESI; [M‐H]‐; PI 38:4MW: 885 ID#: 36 DB: Spec. ListComment: PI 38:4; PI(18:0/20:4); Prec: 885.6; 

LMGP02010003_PE_17_20d5d8d11d14_0 PE 37:4; [M-H]-; GPEtn(17:0/ 20:4(5E,8E,1Head to Tail MF=474 RMF=768

60 120 180 240 300 360 420 480 540 600 660 720 780 840

0

50

100

50

100

196.038106614695

269.24790

303.233382061271

466.29354

466.293757635126606.219070998728 752.511786311441

ID in LipidBlast – First HIT

Name: PE 37:4; [M‐H]‐; GPEtn(17:0/20:4(5E,8E,11E,14E)) ‐ equivalent with GPEtn(17:0/20:4(5Z,8Z,11Z,14Z))MW: 752 ID#: 108245 DB: lipidblast‐negComment: Parent=752.52306 Mz_exact=752.52306 ; PE 37:4; [M‐H]‐; GPEtn(17:0/20:4(5E,8E,11E,14E)); C42H76NO8P

Standard phosphatidylethanolamine – ESI (‐)PE(17:0/20:4(5Z,8Z,11Z,14Z))

Experimental

LipidBlast

LMGP06010006_PI_17_14d9_0017.d, MS PI 31:1; [M-H]-; GPIns(14:1(9Z)/ 17:0)Head to Tail MF=344 RMF=524

60 120 180 240 300 360 420 480 540 600 660 720 780 840

0

50

100

50

100

152.994186408271

225.18534

269.24827916841

361.17814

405.239665818928

541.24153

567.289974906525

793.484186423586

225.18534 999.00 sn1 FA269.24790 999.00 sn2 FA361.17814 400.00 [M‐H]‐sn2‐C6H12O6405.24070 400.00 [M‐H]‐sn1‐C6H12O6523.23097 200.00 [M‐H]‐sn2‐H2O541.24153 999.00 [M‐H]‐sn2567.29353 200.00 [M‐H]‐sn1‐H2O585.30409 999.00 [M‐H]‐sn1

ID in LipidBlast – First HITName: PI 31:1; [M‐H]‐; GPIns(14:1(9Z)/17:0)MW: 793 ID#: 118720 DB: lipidblast‐negComment: Parent=793.48669 Mz_exact=793.48669 ; PI 31:1; [M‐H]‐; GPIns(14:1(9Z)/17:0); C40H75O13P

Standard phosphatidylinositol – ESI(‐)PI(17:0/14:1(9Z))

Experimental

LipidBlast

LMGP06010004_PI_17_20d5d8d11d14_0 PI 37:4; [M-H]-; GPIns(17:0/ 20:4(5E,8E,11EHead to Tail MF=76 RMF=328

70 140 210 280 350 420 490 560 630 700 770 840 910 980

0

50

100

50

100

152.995721701539

269.24790

303.233059154672

405.24077

405.241130531344 567.29459414645

585.30416

871.531958114408

980.490276753085

Experimental

LipidBlast

Standard phosphatidylinositol – ESI(‐)PI(17:0/20:4(5Z,8Z,11Z,14Z))

269.24790 999.00 sn1 FA303.23226 999.00 sn2 FA405.24077 400.00 [M‐H]‐sn2‐C6H12O6439.22513 400.00 [M‐H]‐sn1‐C6H12O6567.29360 200.00 [M‐H]‐sn2‐H2O585.30416 999.00 [M‐H]‐sn2601.27796 200.00 [M‐H]‐sn1‐H2O619.28852 999.00 [M‐H]‐sn1

ID in LipidBlast – First HITName: PI 37:4; [M‐H]‐; GPIns(17:0/20:4(5E,8E,11E,14E))MW: 871 ID#: 119197 DB: lipidblast‐negComment: Parent=871.53368 Mz_exact=871.53368 ; PI 37:4; [M‐H]‐; GPIns(17:0/20:4(5E,8E,11E,14E)); C46H81O13P

Not the END

Hands‐on with course laptops