Autonomous METLIN-Guided In-source Fragment Annotation for

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Autonomous METLIN-guided in-source fragment detection increases annotation confidence in untargeted metabolomics Xavier Domingo-Almenara, J. Rafael Montenegro-Burke, Carlos Guijas, Erica L-W Majumder, H. Paul Benton, and Gary Siuzdak Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03126 • Publication Date (Web): 25 Jan 2019 Downloaded from http://pubs.acs.org on January 26, 2019

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Analytical Chemistry

Autonomous METLIN-guided in-source fragment detection increases annotation confidence in untargeted metabolomics Xavier Domingo-Almenara1,§,∗ , J. Rafael Montenegro-Burke1,§ , Carlos Guijas1,§ , Erica L.-W. Majumder1 , H. Paul Benton1 , Gary Siuzdak1,2 ∗ ∗

To whom correspondence should be addressed: [email protected], [email protected].

1

§

Scripps Center for Metabolomics, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

2

Department of Molecular and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

These authors contributed equally.

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ACS Paragon Plus Environment

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Fig. 1.

In B, the dissociation pathway of methionine at low collision energy is illus-

trated, which shows how multiple fragments can be observed as in-source fragments. In C, MS features obtained after XCMS Online processing are matched against low energy MS/MS spectra (0 and 10 eV) from METLIN. This fragment comparison enables the annotation of these in-source features as in-source fragments (ISF) or FH, protonated/deprotonaded or other ion adduct species, and it provides with a putative identity of the feature based on low energy fragmentation.

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Fig. 2.

Screenshot of MISA’s output. For a given feature selected by the user in

the XCMS Online results table, MISA will output all putative identities. In this example, this feature could be attributed to the protonated species (M+H) of creatine or 3-guanidinopropanoate, since three of their in-source fragments have been observed. The score (match score) and the match (match factor) are two scores computer by MISA used to assess the likelihood of that feature stemming from the metabolite reported by MISA. It could be that this features is in fact an in-source fragment of another molecule, in this case it could be an in-source fragment (ISF) of indolelactic acid. This means that there is another feature in the dataset that has been annotated by MISA as the protonated species of indolelactic acid and this feature has been annotated as its in-source fragment.

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Fig. 3. Among all features associated to identified molecules (datasets 1 and B exhibits the number of neutral masses that were annotated by CAMERA

2), panel

(due to the detection of adducts or common losses), and by MISA (due to in-source annotation). Panel

C shows the total number features stemming from the identified

metabolites (including adducts, common losses and specific ISF) that were annotated by CAMERA, MISA and in total (both methods). Panel

D shows the number of pu-

tative identifications retrieved based on accurate mass search and based on in-source fragment matching; of note, for both methods, only metabolites in METLIN with experimental data were considered. Panels E,F show the overlap between the number of protonated/deprotonated species annotated due to the detection of formed adducts and ISF, in positive and negative mode, respectively, for dataset 3. In

G H the amount

of specific fragments and common losses in positive and negative mode is shown, as percentage of all detected ISF.

DPNQVUFE JG UIF MPXFOFSHZ TQFDUSVN JT DPNQPTFE PG BU MFBTU UISFF GSBHNFOUT "QQMJDBUJPO PG .*4" GPS UIF BOOPUBUJPO PG NFUBCPMJUFT JO VO UBSHFUFE FYQFSJNFOUT 5P JMMVTUSBUF UIF DBQBCJMJUJFT PG UIF QSPQPTFE BQQSPBDI UXP EBUBTFUT UIF ėSTU DPNQSJTJOH NJDF QMBTNB TBNQMFT EBUBTFU BOE UIF TFDPOE DPNQSJTJOH NJDF CSBJO UJTTVF TBNQMFT EBUBTFU XFSF BOBMZ[FE CZ 61-$m.4 JO 31 BOE )*-*$ DISP NBUPHSBQIZ JO QPTJUJWF NPEF 3BX EBUB ėMFT XFSF VQMPBEFE JOUP UIF 9$.4 0OMJOF QMBUGPSN BOE QSPDFTTFE GPS QFBLEFUFDUJPO BOE BMJHO NFOU JO BEEJUJPO UP BOOPUBUJPO WJB $".&Ć BOE UIF .&5-*/ HVJEFE JOTPVSDF BOOPUBUJPO .*4" BMHPSJUIN $".&Ć BOE .*4" XFSF JOEFQFOEFOUMZ BQQMJFE JF $".&Ć XBT VTFE UP BOOP UBUF BEEVDUT BOE DPNNPO MPTTFT XIFSFBT .*4" XBT VTFE UP BOOP UBUF *4' BOE QSPWJEF XJUI B QVUBUJWF NFUBCPMJUF JEFOUJėDBUJPO 0WFS BMM UIF DPNQMFUF QSPDFTTJOH BOE BOOPUBUJPO XPSLĚPX ZJFMEFE UIF EFUFDUJPO PG EBUBTFU BOE EBUBTFU GFBUVSFT JO 31 BOE EBUBTFU BOE EBUBTFU GFBUVSFT JO )*-*$

4

.*4" BTTJHOFE B QVUBUJWF NFUBCPMJUF JEFOUJUZ UP EBUBTFU

BOE EBUBTFU GFBUVSFT JO 31 BOE EBUBTFU BOE EBUBTFU GFBUVSFT JO )*-*$ *EFOUJUJFT PG B UPUBM PG DPNQPVOET BDSPTT EBUBTFUT BOE DISPNBUPHSBQIJD NFUIPET XFSF DPOėSNFE WJB UBOEFN .4 BOE DPNQBSJTPO XJUI TQFDUSBM SFGFSFODF EBUB BOE UIF VTF PG QVSF NBUFSJBMT TFF %BUB 1SPDFTTJOH BOE "OBMZTJT TFDUJPO 5BCMF TIPXT UIF MJTU PG DPSSFDUMZ BOOPUBUFE NFUBCPMJUFT CZ .*4" GPS EBUBTFUT BOE ĉF UBCMF BMTP JODMVEFT UIF SBUJP TDPSF BOE UIF NBUDI GBDUPS UIF OVNCFS PG BEEVDUT SFQPSUFE CZ $".&Ć BOE UIF OVNCFS PG *4' EFUFDUFE CZ .*4" UIBU DPSSFTQPOEFE UP DPN NPO OFVUSBM MPTTFT JO QPTJUJWF JPO NPEF ) 0 /) )$00) BT UIFTF IBWF CFFO TIPXO UP CF UIF NPTU DPNNPO .*4" JODSFBTFT GFBUVSF BOOPUBUJPO QFSGPSNBODF "EEVDUT QMBZ BO JNQPSUBOU SPMF JO QFBL BOOPUBUJPO BT UIFJS PCTFSWBUJPO BMMPXT UIF iUSJBOHVMBUJPOu PG QSPUPOBUFEEFQSPUPOBUFE PS PUIFS JPO BEEVDU TQFDJFT BOE UIFSFGPSF UIF OFVUSBM NBTTFT PG VOEFSMZJOH NFUBCPMJUFT "U UIF TBNF UJNF UIJT BOOPUBUJPO PG QSPUPOBUFE TQFDJFT GBDJMJUBUFT UIF QSFQBSBUJPO PG TVCTFRVFOU UBSHFUFE UBOEFN .4 FYQFSJNFOUT UP JEFOUJGZ UIFTF GFBUVSFT *O DBTFT GSPN B UPUBM PG NFUBCPMJUFT JO EBUBTFUT BOE OP BEEVDUT XFSF GPSNFE PS EFUFDUFEBOOPUBUFE CZ 9$.4$".&Ć 5BCMF ĉJT JNQMJFT UIBU UIF GFBUVSF DPSSF TQPOEJOH UP UIF QSPUPOBUFEEFQSPUPOBUFE TQFDJFT DPVME OPU CF BO OPUBUFE CZ BEEVDUCBTFE BOOPUBUJPO BQQSPBDIFT TVDI BT $".&Ć *OTUFBE .*4" XBT BCMF UP BOOPUBUF B MBSHFS OVNCFS PG QSPUPOBUFE TQFDJFT BOE UIVT OFVUSBM NBTTFT EVF UP UIF QSFTFODF PG *4' 'JHVSF B 4QFDJėDBMMZ B UPUBM PG NFUBCPMJUFT EJTTPDJBUFE JOUP BU MFBTU POF TQFDJėD GSBHNFOU JF OPU B DPNNPO MPTT "MTP BT PCTFSWFE JO 5BCMF POMZ B PG BMM UIF *4' EFUFDUFE CZ .*4" BDSPTT EBUBTFUT

DPSSFTQPOEFE UP DPNNPO OFVUSBM MPTTFT JF MPXTQFDJėDJUZ *4' ĉJT EFNPOTUSBUFT UIBU BEEVDUCBTFE BOOPUBUJPO UPPMT MBDL UIF DBQB CJMJUZ UP BOOPUBUF BO JNQPSUBOU GSBDUJPO PG *4' BT POMZ DPNNPO OFV USBM MPTTFT BT B SFTVMU PG JOTPVSDF GSBHNFOUBUJPO DBO CF BOOPUBUFE WJB BEEVDUCBTFE BOOPUBUJPO ĉJT JT BMTP TVQQPSUFE CZ UIF EBUB TIPXO JO 'JHVSF C XIJDI TIPXT UIBU .*4" BOOPUBUFE UXJDF UIF OVNCFS PG GFBUVSFT DPSSFTQPOEJOH UP UIF JEFOUJėFE NFUBCPMJUFT JO DPNQBS JTPO UP BEEVDUCBTFE BOOPUBUJPO /FYU XF DPNQBSFE UIF OVNCFS PG QVUBUJWF NFUBCPMJUF IJUT CFUXFFO UIF IJUT SFQPSUFE CZ .*4" XJUI UIF IJUT CZ BDDVSBUF NBTT TFBSDI PG UIF PCTFSWFE QSPUPOBUFE TQFDJFT OFVUSBM NBTT 'JHVSF D TIPXT UIBU .*4" XBT BCMF UP SFEVDF UIF MJTU QVUBUJWF JEFOUJUJFT VQ UP B "T EJTDVTTFE JO UIF GPMMPXJOH TFD UJPO NBOVBM DVSBUJPO VTJOH UIF .*4" BOOPUBUJPO TDPSFT DBO CF VTFE UP GVSUIFS SFEVDF UIF MJTU BOE QVUBUJWF NFUBCPMJUFT %VF UP MPX JOUFOTJUZ QFBLT BOE OPJTF BEEVDUT BOE DPNNPO MPTTFT NJHIU OPU CF EFUFDUFE CZ 9$.4 PS BOOPUBUFE CZ $".&Ć 5P EFNPOTUSBUF UIF BEWBOUBHF PG PVS BQQSPBDI XF BEEJUJPOBMMZ FWBMV BUFE UIF BMHPSJUIN UISPVHI UIF BOOPUBUJPO PG IVNBO VSJOF TBNQMFT BOBMZ[FE CZ 61-$ JO 31 DPVQMFE UP B MJOFBS RVBESVQPMF JPO USBQ 0SCJUSBQ .4 JO CPUI QPTJUJWF BOE OFHBUJWF NPEF EBUBTFU QSF WJPVTMZ QVCMJTIFE FMTFXIFSF ĉJT EBUBTFU VOEFSXFOU B NBOVBM JOTQFDUJPO PG QFBLT BOE DPNNPO BEEVDUT BOE MPTTFT UIBU XFSF OPU EFUFDUFE CZ 9$.4 PS BOOPUBUFE CZ $".&Ć XFSF TFBSDIFE JO UIF SBX EBUB 'SPN BMM UIF NFUBCPMJUFT JEFOUJėFE JO UIF PSJHJOBM TUVEZ B UPUBM PG QPTJUJWF NPEF BOE OFHBUJWF NPEF IBE FY QFSJNFOUBM .4.4 EBUB JO .&5-*/ 'SPN UIPTF .*4"T QVUBUJWF JEFOUJėDBUJPO SFQPSUFE UIF DPSSFDU JEFOUJUZ GPS BOE

NFUBCPMJUFT CBTFE PO *4' 0O UIF PUIFS IBOE PG BOE


Page 4 of 9

Page 5 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 4.


Examples of spectral similarity between MS pseudo-spectra (black) and low energy MS/MS spectra extracted from METLIN (color, negatively rotated), for eight

metabolites. Protonated ion/precursor (*) together with MS/MS collision energy (CE) are noted. Low energy MS/MS spectra at 5 eV were calculated by fragment intensity interpolation between 0 and 10 eV.

PG GSPN BMM UIF NFUBCPMJUFT XJUI TQFDUSBM EBUB JO .&5-*/ GPSNFE DPNNPO BEEVDUT , /B $M FUD PS EJNFST 'JHVSF E F *U JT JNQPSUBOU UP IJHIMJHIU UIBU TPNF NPMFDVMFT EJE OPU GPSN BOZ BEEVDU CVU EJTTPDJBUFE JOUP *4' BOE WJDF WFSTB 'JHVSF E F ĉJT EFNPOTUSBUFT UIBU FBDI UZQF PG BOOPUBUJPO *4'CBTFE BOE BEEVDUCBTFE QSPWJEF B DPNQMFNFOUBSZ BOOPUBUJPO DPWFSBHF BOE UIBU UIF DPNCJOBUJPO PG BEEVDU BOE JOTPVSDF GSBHNFOU BOOPUBUJPO JT NPSF FĎFDUJWF UIBO USBEJUJPOBM DPNQVUBUJPOBM BOOPUBUJPO TUSBUF HJFT SFMZJOH PO BEEVDUT BOE DPNNPO OFVUSBM MPTTFT BMPOF 'JOBMMZ NPSF UIBO BOE PG GSBHNFOUT EFUFDUFE CZ PVS BQ QSPBDI DPSSFTQPOEFE UP TQFDJėD *4' PG FBDI NFUBCPMJUF XIFSFBT UIF SFNBJOJOH DPSSFTQPOEFE UP DPNNPO OFVUSBM MPTTFT 'JHVSF G H 3PCVTU NFUBCPMJUF BOOPUBUJPO CBTFE PO MPXFOFSHZ ěBHNFOUBUJPO "T NFOUJPOFE CFGPSF UIF DPNCJOFE VTF PG BEEVDU BOE JOTPVSDF BOOPUBUJPO BMMPXFE VODPWFSJOH B MBSHFS OVNCFS PG OFVUSBM NBTTFT DPNQBSFE UP BEEVDU BOOPUBUJPO BMPOF ĉFTF OFVUSBM NBTTFT DPVME CF TFBSDIFE BHBJOTU EBUBCBTFT UP ėOE QVUBUJWF NFUBCPMJUF JEFOUJė DBUJPOT )PXFWFS UIJT JT DPOTJEFSFE BT B XFBL NFUBCPMJUF BOOPUB UJPO NFUIPE *OTUFBE .*4" QSPWJEFT XJUI SPCVTU QVUBUJWF JEFO UJėDBUJPOT CBTFE PO JOTPVSDF GSBHNFOU NBUDIJOH DPOTJTUJOH PG UIF OBNF PG UIF DBOEJEBUF XJUI UXP NBUDIJOH TDPSFT OBNFMZ SBUJP TDPSF BOE NBUDI GBDUPS TFF $PNQVUBUJPOBM XPSLĚPX ĉF SBUJP TDPSF SF QPSUFE CZ .*4" OVNCFS PG GSBHNFOUT GPVOE PVU PG UIF UPUBM OVN CFS PG GSBHNFOUT JO MPX FOFSHZ TQFDUSB JO .&5-*/ JT HJWFO BT B GSBDUJPO BOE JU TFSWFT BT BO JOEJDBUPS PG IPX SFMJBCMF QVUBUJWF JEFO UJėDBUJPOT BSF *OUVJUJWFMZ UIFSF JT B IJHI MJLFMJIPPE UIBU B QVUBUJWF JEFOUJUZ JT DPSSFDU JO DBTFT XIFSF B NPMFDVMF EJTTPDJBUFT JOUP NVM

UJQMF GSBHNFOUT BOE UIF NBKPSJUZ PG UIPTF GSBHNFOUT JO MPX FOFSHZ TQFDUSB BSF GPVOE JO .4 EBUB FH USZQUPQIBO

DJUSVMMJOF

/"% PS PYJEJ[FE HMVUBUIJPOF JO 5BCMF ĉJT JT BMTP TVQQPSUFE CZ UIF GBMTF EJTDPWFSZ SBUF '%3 EFDSFBTF BU IJHI TDPSF SBUJPT TFF 4VQQMFNFOUBSZ 'JHVSF 4 BOE 4VQQMFNFOUBSZ %BUB GPS EFUBJMT PO UIF '%3 DBMDVMBUJPO )PXFWFS OPU BMM NFUBCP MJUFT SFBEJMZ EJTTPDJBUF JOUP NVMUJQMF GSBHNFOUT BOE UIFTF BSF VTVBMMZ MPXJOUFOTJUZ QFBLT UIBU DBO CF FBTJMZ NBTLFE CZ OPJTF 'PS BMM UIFTF SFBTPOT JU JT OPU QPTTJCMF UP FTUBCMJTI B TDPSF UISFTIPME UP DPOTJEFS B QVUBUJWF JEFOUJėDBUJPO BT DPSSFDU BOE BMM UFOUBUJWF JEFOUJėDBUJPOT IBWF UP CF DPOėSNFE WJB UBOEFN .4 /FYU XF GPDVTFE PO JOUFOTJUZ TJNJMBSJUJFT CFUXFFO *4' BOE MPX FOFSHZ .4.4 TQFDUSB ĉFTF JOUFOTJUZ TJNJMBSJUJFT BSF BTTFTTFE CZ UIF NBUDI GBDUPS TDPSF DBMDVMBUFE CZ .*4" 'JHVSF TIPXT FYBN QMFT PG DPSSFDU JEFOUJėDBUJPOT QSPWJEFE CZ .*4" PG DPNQPVOET XJUI NVMUJQMF *4' UPHFUIFS XJUI UIFJS DPSSFTQPOEJOH MPX FOFSHZ .4.4 TQFDUSB 4PNF NFUBCPMJUFT MJLF UZSPTJOF DJUSVMMJOF PS MJOPMFJD BDJE SFBEJMZ EJTTPDJBUF JO UIF TPVSDF JOUP NVMUJQMF GSBHNFOUT "T PCTFSWFE JO UIF ėHVSF GSBHNFOU SFMBUJWF JOUFOTJUJFT CFUXFFO .4.4 MPX FO FSHZ TQFDUSB F7 PS F7 BOE .4 QTFVEPTQFDUSB DBO WBSZ *O UIFTF FYBNQMFT NPTU *4' BSF PCTFSWFE JO UIF F7 TQFDUSB 8IJMF SFMBUJWF JOUFOTJUJFT BSF TJNJMBS JO TPNF DBTFT TQIJOHPTJOF PYJEJ[FE HMVUBUIJPOF QIPTQIPDIPMJOF BOE UIJBNJOF

UIFTF BSF OPU TJNJMBS GPS PUIFS NFUBCPMJUFT DJUSVMMJOF *U NJHIU PDDVS UIBU UIF CFTU JOUFO TJUZ TJNJMBSJUZ JT BU F7 DPVNBSJD BDJE 0UIFS NFUBCPMJUFT TVDI BT UZSPTJOF BOE MJOPMFJD BDJE TIPX UIF CFTU TJNJMBSJUZ BU F7 GPS JMMVT USBUJWF QVSQPTFT XF DPNQVUFE UIF F7 TQFDUSB CZ JOUFOTJUZ JOUFSQP


5


/P 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

Table 1. List of correctly annotated %BUBTFU /BNF 3BUJP 4 /P" /PDocosahexaenoic acid 41/94 5 1 Oleic Acid 35/84 4 1 Palmitic acid 22/49 5 Methionine 4/12 2 Myristic acid 13/44 4 Linoleic acid 63/100 5 1 Arachidonic Acid 8/58 4 1 Myristoleic acid 1/53 2-Linoleoyl Glycerol 11/66 3 1 Proline 1/2 1 Leucine 1/2 1 Glutamine 3/7 1 Histidine 1/7 1 Phenylalanine 2/7 1 Ectoine 1/8 1 Methionine 6/14 2 Hippuric acid 1/11 4-Hydroxyisoleucine 3/16 1 Ornithine 3/15 3 2 Arginine 4/10 5 2 Methionine S-oxide 4/19 3 N-Acetyl-L-lysine 2/6 2 1 Isoleucine 1/4 1 Tryptophan 10/12 2 Glutamine 5/8 5 2 Carnitine 2/9 Propionylcarnitine 1/13 Citrulline 9/12 3 2 Leucine 1/3 1 Phenylalanine 5/7 2 Acetylcarnitine 3/6 3 Kojibiose 4/40 Cystine 3/41 2 1 Histidine 4/7 1 Acetyl-DL-Leucine 4/7 2 Glycerophosphocholine 2/6 3 5-L-Glutamyl-L-alanine 3/12 1 trans-Cinnamic acid 4/29 2 Asparagine 3/13 2 LysoPC(16:0) 2/3 1 Theanine 4/17 2 Lysine 3/14 2 Tyrosine 7/14 2 Gamma-glutamylglycine 2/14 1 Creatine 3/9 2

metabolites by MISA for Datasets 1 and 2.a %BUBTFU .' /BNF 3BUJP 4 /P" 87 Diethyltoluamide 1/5 66 Methionine 7/13 43 Sphinganine 1/12 1 85 Guanidylic acid 3/7 3 16 Sphingosine 4/9 2 77 1-AG 6/61 67 Guanine 2/3 1 Trigonelline 1/7 87 Phosphocholine 3/5 2 Betaine 1/2 NADP+ 6/10 79 NAD 9/14 2 100 Tryptophan 7/12 100 Arginine 9/10 5 99 Argininosuccinic acid 1/16 2 81 Citrulline 4/12 100 Tryptophan 8/12 17 N-Acetyl-D-phenylalanine 4/17 39 NAD 7/14 2 35 Glutathione, oxidized 10/16 22 Cystine 1/39 92 Cystathionine 4/13 99 Guanidylic acid 2/7 9 99 Lysine 5/14 3 67 Histidine 4/7 71 Carnosine 11/28 6 28 Phosphocholine 2/5 9 95 Methionine S-oxide 3/18 3 100 Threonine 2/10 100 Taurine 1/4 52 Thiamine 3/5 75 N-Methylglutamic acid 3/6 72 N2-Acetyl-L-ornithine 3/17 100 Glutamic acid 5/5 51 DL-o-Tyrosine 6/9 100 m-Coumaric acid 5/6 30 Sphingosine-1-phosphate 4/36 59 40 64 10 36 96 44 100

Page 6 of 9

/P-

2 1 1 1 1

2 2 1 1 2 1 1 1 3 1 3 1 1 1 2 3 2 3

.' 100 21 94 71 100 70 85 71 88

13 6 100 87 43 98 99 87 58 87 84 100 100 57 100 59 98 90 97 99 99 71 26 84 75 78 2

a .FUBCPMJUF JEFOUJUJFT UPHFUIFS XJUI UIFJS .*4" SBUJP TDPSF 3BUJP 4 DPNQVUFE BT UIF OVNCFS PG GSBHNFOUT GPVOE PVU PG UIF UPUBM OVNCFS PG GSBHNFOUT JO

UIF MPX FOFSHZ TQFDUSB ĉF OVNCFS PG BEEVDUT SFQPSUFE CZ $".&Ć /P" BOE UIF OVNCFS PG GSBHNFOUT UIBU DPSSFTQPOE UP DPNNPO OFVUSBM MPTTFT /P - BSF BMTP OPUFE ĉF NBUDI GBDUPS .' EFOPUJOH UIF TQFDUSBM TJNJMBSJUZ JT BMTP TIPXO .FUBCPMJUF OBNFT BQQFBS EVQMJDBUFE JO TPNF DBTFT BT UIFZ IBWF CFFO EFUFDUFE JO CPUI 31 BOE )*-*$ "MTP JO TPNF DBTFT UIF TBNF NFUBCPMJUF JO B EJĎFSFOU NPEF 31)*-*$ IBWF B EJĎFSFOU UPUBM OVNCFS PG GSBHNFOUT 3BUJP 4 ĉJT JT CFDBVTF .*4" EFUFSNJOFT UIF UPUBM OVNCFS PG GSBHNFOUT XJUIJO UIF .4 BDRVJTJUJPO SBOHF ĉFSFGPSF JG B .4 JT DPOėHVSFE UP BDRVJSF JO B OBSSPXFS SBOHF XIJDI EPFT OPU DPWFS UIF FOUJSF MPX FOFSHZ .4.4 SBOHF B TNBMMFS UPUBM OVNCFS PG GSBHNFOUT XJMM CF SFQPSUFE JO UIF SBUJP TDPSF

MBUJPO CFUXFFO BOE F7 ĉFTF TJNJMBSJUJFT XJMM EFQFOE PO FY QFSJNFOUBM DPOEJUJPOT BOE JOTUSVNFOUT $PFMVUJPO PG DPNQPVOET

6

XJUI TJNJMBS TUSVDUVSFT NBTT TFMFDUJWF USBOTNJTTJPO &4* OFFEMF BOE MFOT WPMUBHFT PS UFNQFSBUVSF DBO BMTP BĎFDU SFMBUJWF JOUFOTJUJFT %FTQJUF UIF WBSJBCJMJUZ PG SFMBUJWF JOUFOTJUJFT UIF NBUDI GBDUPS QSP WJEFE CZ .*4" DBO CF VTFE UP SBOL DBOEJEBUFT TIBSJOH UIF TBNF GSBH NFOUT 4JNJMBS DPNQPVOET MJLF HMVUBNJD BDJE BDFUZMTFSJOF BOE


Page 7 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 5.


For a given feature, three putative identities, glutamic acid, acetylserine and 3-methylaspartic acid are reported by MISA based on in-source fragment matching.

As their chemical structures are similar, they share common fragments (a). Despite being glutamic acid the correct identity (C), false positives might be also reported (D, E). The match factor (MF) can be used to asses spectral similarity.

NFUIZMBTQBSUJD BDJE DPOTJEFSBCMZ EJTTPDJBUF BU MPX FOFSHZ BOE BU UIF TBNF UJNF UIFZ TIBSF DPNNPO GSBHNFOUT 'JHVSF ĉFTF UISFF NFUBCPMJUFT XJMM IBWF EJĎFSFOU FMVUJPO UJNFT IPXFWFS XJUIPVU UIF BOBMZTJT PG QVSF TUBOEBSET UIF SFUFOUJPO UJNF SFNBJOT VOLOPXO BOE DBOOPU CF UBLFO JOUP BDDPVOU CZ .*4" *O UIFTF DBTFT .*4" XJMM SF QPSU NVMUJQMF DBOEJEBUFT QFS GFBUVSF BT QFBLT GPVOE JO .4 NJHIU PSJHJOBUF GSPN BOZ PG UIPTF UISFF NFUBCPMJUFT *O UIFTF DBTFT UIF NBUDI GBDUPS DBO CF VTFE UP SBOL UIF DBOEJEBUFT "T BO FYBNQMF 'JH VSF TIPXT HMVUBNJD BDJE IBT B IJHI TJNJMBSJUZ CFUXFFO JUT *4' BOE UIF MPX FOFSHZ TQFDUSVN XIFSFBT UIF PUIFS UXP DBOEJEBUFT BDFUZMTFSJOF BOE NFUIZMBTQBSUJD BDJE TIPX B MPX TJNJMBSJUZ BOE MPX NBUDI GBDUPS WBMVFT BOE SFTQFDUJWFMZ (MVUBNJD BDJE XBT DPOėSNFE BT UIF DPSSFDU JEFOUJUZ VTJOH .4.4 PO UIF BOOPUBUFE GFBUVSF *U JT XPSUI OPUJOH UIBU UIF NBUDI GBDUPS NFBTVSFT TQFDUSBM TJNJMBSJUZ BOE JO FĎFDU JU QFSGPSNT B DPSSFMBUJPO CFUXFFO *4' BOE MPXFOFSHZ TQFDUSB *U JT XFMM LOPXO UIBU UIF DPSSFMBUJPO SFMJBCJMJUZ EFQFOET PO IPX NBOZ EBUB QPJOUT BSF VTFE JF IPX NBOZ *4' BSF CFJOH DPNQBSFE ĉJT NJHIU MFBE UP OPOTJHOJėDBOU NBUDI GBDUPST JO DBTFT XIFSF NPMFDVMFT EJTTPDJBUF JOUP B GFX GSBHNFOUT BU MPX FO FSHZ PS GFX *4' BSF EFUFDUFE &YBNQMFT PG UIJT JODMVEF JTPMFVDJOF PS MFVDJOF 5BCMF SPX OVNCFS BOE

UIBU EJTTPDJBUF JOUP UISFF

GSBHNFOUT BU MPX FOFSHZ 0UIFS DBTFT JODMVEF IJTUJEJOF PS QIFOZMBMB OJOF 5BCMF SPX OVNCFS BOE

XIFSF EFTQJUF UIBU UIFZ EJT TPDJBUF JOUP NVMUJQMF GSBHNFOUT POMZ POF PS UXP *4' XFSF EFUFDUFE TJODF UIF SFNBJOJOH GSBHNFOUT JO UIF MPX FOFSHZ TQFDUSVN BSF MPX JOUFOTJUZ GSBHNFOUT ĉJT JT XIZ JU JT JNQPSUBOU UP VTF UIF NBUDI GBD UPS JO DPNCJOBUJPO XJUI UIF SBUJP TDPSF BT UIF SBUJP TDPSF JOEJDBUFT UIF OVNCFS PG GSBHNFOUT JOWPMWFE JO UIF NBUDI GBDUPS DPNQVUBUJPO 4PNF MJNJUBUJPOT PG .*4" JODMVEF UIF SFQPSU PG GBMTF QPTJUJWF QV UBUJWF JEFOUJėDBUJPOT &YBNQMFT PG GBMTF QPTJUJWFT SFQPSUFE CZ .*4" BSF TIPXO JO 5BCMF XIFSF UIF DPSSFDU JEFOUJėDBUJPOT UPHFUIFS XJUI GBMTF QPTJUJWF JEFOUJėDBUJPOT BSF SFQPSUFE GPS UIF TBNF GFBUVSF 8IFO UIF TBNF *4' BSF TIBSFE CZ EJĎFSFOU NPMFDVMFT BMM UIFTF NPMFDVMFT BSF HPJOH UP CF SFQPSUFE BT QVUBUJWF JEFOUJėDBUJPOT *O NBOZ DBTFT UIFTF NPMFDVMFT XJMM CF TJNJMBS PS XJMM TIBSF B TVCTUSVDUVSF ĉJT DBO CF JMMVTUSBUFE BMTP XJUI UIF TBNF FYBNQMF TIPXO JO 'JHVSF XIFSF TJNJMBS DPNQPVOET MJLF HMVUBNJD BDJE BDFUZMTFSJOF BOE NFUIZMBTQBSUJD BDJE BSF SFQPSUFE BT QPUFOUJBM JEFOUJUZ DBOEJEBUFT GPS UIF PCTFSWFE GFBUVSF 'PMMPXJOH UIF TBNF FYBNQMF JU DPVME BMTP PDDVS UIBU OPOF PG UIF DBOEJEBUFT QSPQPTFE CZ .*4" DPSSFTQPOET UP UIF USVF NFUBCPMJUF JEFOUJUZ ĉJT XJMM PDDVS XIFO B NPMFDVMF UIBU MBDLT PG FYQFSJNFOUBM TQFDUSBM EBUB JO .&5-*/ JT CFJOH EF


7


/P 1 2

3 4

5

Table 2. Examples of false positive identifications.b 5SVF .PMFDVMF 'BMTF 1PTJUJWF Creatine (3/9 - 100%) 3-Guanidinopropanoate L-Theanine (4/17 - 10%) N2-Acetyl-L-ornithine Acetylproline (M+NH4 ) Acetylcarnitine (3/6 - 52%) Acetyl-L-carnitine hydrochloride Isoleucine (1/4 - 99%) Leucine Norleucine Alloisoleucine N-Methylglutamic acid (3/6 - 71%) 2-Aminoadipic acid

Page 8 of 9

4DPSF 3/16 - 42% 3/17 - 8% 3/18 - 7% 3/5 - 39% 1/3 - 100% 1/4 - 100% 1/5 - 99% 5/8 - 53%

b *O DBTFT XIFSF NVMUJQMF NFUBCPMJUFT TIBSF DPNNPO GSBHNFOUT .*4" XJMM SFQPSU CPUI UIF USVF BOE GBMTF QPTJUJWF JEFOUJUJUFT

*O UIFTF FYBNQMFT UIF USVF NPMFDVMF UPHFUIFS XJUI UIFJS SBUJP TDPSF GSBDUJPO BOE NBUDI GBDUPS QFSDFOUBHF BSF MJTUFE 'PS FBDI USVF JEFOUJUZ UIF UBCMF JOEJDBUFT UIF GBMTF QPTJUJWFT SFQPSUFE CZ .*4" BMTP XJUI UIFJS SFTQFDUJWF TDPSFT

UFDUFE BOE TIBSFT GSBHNFOUT XJUI TJNJMBS NPMFDVMFT JO .&5-*/ FH JG HMVUBNJD BDJE TQFDUSB XFSF OPU JODMVEFE JO .&5-*/ .*4" XPVME POMZ SFQPSU BDFUZMTFSJOF BOE NFUIZMBTQBSUJD BDJE BT QVUB UJWF DBOEJEBUFT *O UIJT DBTF UIF EFUFDUJPO PG GPVS EJTUJODUJWF GSBH NFOUT TIBSFE CZ UIFTF NPMFDVMFT XPVME IBWF TVHHFTUFE UIBU UIF USVF NPMFDVMF TIBSFE B DFSUBJO EFHSFF PG TJNJMBSJUZ PS B DPNNPO TVCTUSVD UVSF XJUI NPMFDVMFT SFQPSUFE CZ .*4" "OPUIFS TDFOBSJP XIFSF GBMTF QPTJUJWFT BSF MJLFMZ UP PDDVS JT EVSJOH TIPSU -$ HSBEJFOUT *O TVDI DBTFT DPFMVUJPO PG EJĎFSFOU NPMFDVMFT XPVME JODSFBTF CPUI UIF OVNCFS PG GFBUVSFT BOE QPTTJCMF *4' JODSFBTJOH UIF NBUDI QPTTJCJM JUJFT 0WFSBMM BDDPSEJOH UP UIF HVJEFMJOFT CZ UIF .FUBCPMPNJDT 4UBO EBSET *OJUJBUJWF .4* .*4" DBO CF VTFE UP SFUSJFWF MFWFM QV UBUJWF JEFOUJėDBUJPOT JO DBTFT XIFSF EJBHOPTUJD GSBHNFOUT PS OFVUSBM MPTTFT DPOTJTUFOU XJUI B TQFDJėD TUSVDUVSF BSF PCTFSWFE .PSFPWFS EFTQJUF UIF GBDU UIBU .&5-*/ JT POF PG UIF MBSHFTU MJCSBSJFT PG FYQFS JNFOUBM .4.4 TQFDUSB BO FYUFOTJWF OVNCFS PG NFUBCPMJUFT MBDL PG FYQFSJNFOUBM TQFDUSB 'PS UIFTF SFBTPOT .*4" QVUBUJWF JEFOUJ ėDBUJPOT SFRVJSF B MFWFM DPOėSNBUJPO VTJOH UBOEFN .4 VTJOH TUBOEBSE NBUFSJBMT JO DPNCJOBUJPO XJUI PSUIPHPOBM UFDIOJRVFT VO EFS JEFOUJDBM BOBMZUJDBM DPOEJUJPOT

CONCLUSIONS %FTQJUF UIF FYJTUFODF PG DPNQVUBUJPOBM BOOPUBUJPO BMHPSJUINT B MBSHF OVNCFS PG GFBUVSFT PCTFSWFE JO VOUBSHFUFE NFUBCPMPNJDT QSP ėMJOH TUJMM SFNBJO VOBOOPUBUFE .PSF UIBO PG NPMFDVMFT JO DMVEFE JO TQFDUSBM EBUBCBTFT SFBEJMZ EJTTPDJBUF JOUP NVMUJQMF GSBH NFOUT BU MPX DPMMJTJPO FOFSHZ XIJDI JNQMJFT UIBU B DPOTJEFSBCMF GSBDUJPO PG UIPTF GFBUVSFT DPSSFTQPOET UP JOTPVSDF GSBHNFOUT )FSF XF QSFTFOU B TUSBUFHZ UP BOOPUBUF JOTPVSDF GSBHNFOUT WJB DPNQBSJ TPO XJUI .&5-*/T FYQFSJNFOUBM MPX FOFSHZ .4.4 TQFDUSB 8F EFNPOTUSBUFE UIBU UIF DPNCJOFE VTF PG BEEVDU BOE JOTPVSDF BOOP UBUJPO BMMPXFE VODPWFSJOH B MBSHFS OVNCFS PG OFVUSBM NBTTFT DPN QBSFE UP BEEVDU BOOPUBUJPO BMPOF .PSF JNQPSUBOUMZ .*4" FOBCMFE UIF BOOPUBUJPO PG OFVUSBM NBTTFT FWFO XIFO BEEVDUT XFSF OPU PC TFSWFE "U UIF TBNF UJNF .*4" FOBCMFE B SPCVTU QVUBUJWF NFUBCP MJUF BOOPUBUJPO CBTFE PO MPX FOFSHZ TQFDUSBM DPNQBSJTPO ĉFTF BO OPUBUJPOT DBO CF VTFE UP SFEVDF UIF OVNCFS PG QPTTJCMF NFUBCPMJUF DBOEJEBUFT UIBU DBO CF BĨSJCVUFE UP B GFBUVSF 0WFSBMM UIF QSFTFOUFE TUSBUFHZ QFSGPSNT B DPNQMFNFOUBSZ BOOP UBUJPO MBZFS CZ MFWFSBHJOH UIF JOGPSNBUJPO GSPN FYQFSJNFOUBM MPX 8

FOFSHZ TQFDUSB JO .&5-*/ DVSSFOUMZ DPWFSJOH NPSF UIBO NPMFDVMFT ĉJT TUSBUFHZ BMMPXT JOTPVSDF GSBHNFOUT UP CF BOOPUBUFE JODSFBTJOH JO UVSO NFUBCPMJUF BOOPUBUJPO DPOėEFODF 6MUJNBUFMZ PVS BVUPOPNPVT TUSBUFHZ DPOUSJCVUFT UP TUSFBNMJOJOH GFBUVSF BOOP UBUJPO BOE JEFOUJėDBUJPO JO VOUBSHFUFE NFUBCPMPNJDT ACKNOWLEDGMENTS. ĉJT SFTFBSDI XBT QBSUJBMMZ GVOEFE CZ &DPTZTUFNT BOE /FUXPSLT *OUFHSBUFE XJUI (FOFT BOE .PMFDVMBS "TTFNCMJFT &/*(."

B 4DJFOUJėD 'PDVT "SFB 1SP HSBN BU -BXSFODF #FSLFMFZ /BUJPOBM -BCPSBUPSZ GPS UIF 64 %FQBSUNFOU PG &OFSHZ 0đDF PG 4DJFODF 0đDF PG #JPMPHJDBM BOE &OWJSPONFOUBM 3FTFBSDI VOEFS DPOUSBDU OVNCFS %&"$ $) BOE /BUJPOBM *OTUJUVUFT PG )FBMUI HSBOUT 3 (. 1 .) BOE 1 %" Code availability: ĉF GVMM JNQMFNFOUBUJPO PG .*4" JT JOUFHSBUFE JO IĨQYDNTPOMJOFTDSJQQTFEV ĉF TPVSDF DPEF GPS EFWFMPQFST JT BWBJMBCMF BU IĨQTHJUIVCDPNYEPNJOHPBMNJTB3 Supporting Information Available: .FUIPET UP EFUFSNJOF UIF USVF BOE GBMTF QPTJUJWF '1

EJTUSJCVUJPOT BOE GBMTF EJTDPWFSZ SBUF '%3 GPS CPUI SBUJP BOE EPU QSPEVDU TDPSFT UPHFUIFS XJUI UIF SFTQFDUJWF ėHVSF TIPXJOH UIF '1 EJTUSJCVUJPO BOE '%3

REFERENCES. $IBMFDLJT 3 .FJTUFS * ;IBOH 1 8IFFMPDL $ & $IBMMFOHFT QSPHSFTT BOE QSPNJTFT PG NFUBCPMJUF BOOPUBUJPO GPS -$m.4CBTFE NFUBCPMPNJDT $VSS 0QJO #JPUFDIOPM m 7JOBJYB . 4BNJOP 4 4BF[ * %VSBO + (VJOPWBSU + + :BOFT 0 " (VJEFMJOF UP 6OJWBSJ BUF 4UBUJTUJDBM "OBMZTJT GPS -$.4#BTFE 6OUBSHFUFE .FUBCPMPNJDT%FSJWFE %BUB .FUBCP MJUFT m .BIJFV / ( 4QBMEJOH + - (FMNBO 4 + 1BĨJ ( + %FėOJOH BOE %FUFDUJOH $PNQMFY 1FBL 3FMBUJPOTIJQT JO .BTT 4QFDUSBM %BUB ĉF .[VOJUZ "MHPSJUIN "OBM $IFN m 9V : ' -V 8 3BCJOPXJU[ + % "WPJEJOH NJTBOOPUBUJPO PG JOTPVSDF GSBHNFOUB UJPO QSPEVDUT BT DFMMVMBS NFUBCPMJUFT JO MJRVJE DISPNBUPHSBQIZNBTT TQFDUSPNFUSZCBTFE NFUBCPMPNJDT "OBM $IFN m 4QJDFS 3 4BMFL 3 ..PSFOP 1 $BÒVFUP % 4UFJOCFDL $ /BWJHBUJOH GSFFMZBWBJMBCMF TPěXBSF UPPMT GPS NFUBCPMPNJDT BOBMZTJT .FUBCPMPNJDT %PNJOHP"MNFOBSB 9 FU BM "OOPUBUJPO " $PNQVUBUJPOBM 4PMVUJPO GPS 4USFBNMJOJOH .FUBCPMPNJDT "OBMZTJT "OBM $IFN m .JTSB # # /FX UPPMT BOE SFTPVSDFT JO NFUBCPMPNJDT m &MFDUSPQIPSFTJT m -ZOO , 4 $IFOH . - $IFO : 3 )TV $ $IFO " -JI 5 . $IBOH ) : )VBOH $ + 4IJBP . 4 1BO 8 ) 4VOH 5 : )TV 8 - .FUBCPMJUF JEFOUJėDBUJPO GPS NBTT TQFDUSPNFUSZCBTFE NFUBCPMPNJDT VTJOH NVMUJQMF UZQFT PG DPSSFMBUFE JPO JOGPSNBUJPO "OBM $IFN m #SPFDLMJOH $ % (BOOB " -BZFS . #SPXO , 4VĨPO # *OHFMTTPO & 1FFST ( 1SFOOJ + & &OBCMJOH &đDJFOU BOE $POėEFOU "OOPUBUJPO PG -$¢.4 .FUBCPMPNJDT %BUB UISPVHI .4 4QFDUSVN BOE 5JNF 1SFEJDUJPO "OBM $IFN m .POUFOFHSP#VSLF + 3 4VĨPO + " 3PHFST - . .JMOF ( - .D-FBO + " "SPOPĎ % . -JQJE QSPėMJOH PG QPMBSJ[FE IVNBO NPOPDZUFEFSJWFE NBDSPQIBHFT 1SPTUBHMBOEJOT 0UIFS -JQJE .FEJBU m ;BNėS " 1FUFS଑,BUBMJOJˀ + $BQJMMBSZ FMFDUSPQIPSFTJT଑NBTT TQFDUSPNFUSZ GPS HMZDPTDSFFO JOH JO CJPNFEJDBM SFTFBSDI &MFDUSPQIPSFTJT m %PNJOHP"MNFOBSB 9 #SF[NFT + 7JOBJYB . 4BNJOP 4 3BNJSF[ / 3BNPO,SBVFM . -FSJO $ %ÎB[ . *CÃÒF[ - $PSSFJH 9 1FSFSB-MVOB " :BOFT 0 F3BI " $PN QVUBUJPOBM 5PPM *OUFHSBUJOH 4QFDUSBM %FDPOWPMVUJPO BOE "MJHONFOU XJUI 2VBOUJėDBUJPO BOE *EFOUJėDBUJPO PG .FUBCPMJUFT JO ($.4#BTFE .FUBCPMPNJDT "OBM $IFN m 'FOO + # "OHFX $IFN *OU &E &OHM m (VJKBT $ FU BM .&5-*/ " 5FDIOPMPHZ 1MBUGPSN GPS *EFOUJGZJOH ,OPXOT BOE 6OLOPXOT "OBM $IFN m


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m 8BDITNVUI $ + 7PHM ' $ 0FGOFS 1 + %FĨNFS , $ISPNBUPHSBQIJD .FUIPET JO .FUBCPMPNJDT ĉF 3PZBM 4PDJFUZ PG $IFNJTUSZ ,JOE 5 'JFIO 0 .FUBCPMPNJD EBUBCBTF BOOPUBUJPOT WJB RVFSZ PG FMFNFOUBM DPNQPTJUJPOT NBTT BDDVSBDZ JT JOTVđDJFOU FWFO BU MFTT UIBO QQN #.$ #JPJOG

4VNOFS - 8 "NCFSH " #BSSFĨ % #FBMF . ) #FHFS 3 %BZLJO $ " 'BO 5 8 'JFIO 0 (PPEBDSF 3 (SJđO + - )BOLFNFJFS 5 )BSEZ / )BSOMZ + )JHBTIJ 3 ,PQLB + -BOF " / -JOEPO + $ .BSSJPĨ 1 /JDIPMMT " 8 3FJMZ . % ĉBEFO + + 7JBOU . 3 1SPQPTFE NJOJNVN SFQPSUJOH TUBOEBSET GPS DIFNJDBM BOBMZTJT $IFNJDBM "OBMZTJT 8PSLJOH (SPVQ $"8( .FUBCPMPNJDT 4UBOEBSET *OJUJBUJWF .4* .FUBCPMPNJDT m 4DISJNQF3VUMFEHF "$ $PESFBOV 4( 4IFSSPE 4% .D-FBO +" 6OUBSHFUFE .FUBCPMPNJDT 4USBUFHJFTã$IBMMFOHFT BOE &NFSHJOH %JSFDUJPOT + "N 4PD .BTT 4QFDUSPN 7JOBJYB . 4DIZNBOTLJ & - /FVNBOO 4 /BWBSSP . 4BMFL 3 . :BOFT 0 .BTT TQFDUSBM EBUBCBTFT GPS -$.4 BOE ($.4CBTFE NFUBCPMPNJDT 4UBUF PG UIF ėFME BOE GV UVSF QSPTQFDUT 5SFOET "OBMZU $IFN m 'SBJOBZ $ 4DIZNBOTLJ &- /FVNBOO 4 .FSMFU # 4BMFL 3. +PVSEBO ' :BOFT 0 .JOE UIF (BQ .BQQJOH .BTT 4QFDUSBM %BUBCBTFT JO (FOPNF4DBMF .FUBCPMJD /FUXPSLT 3FWFBMT 1PPSMZ $PWFSFE "SFBT .FUBCPMJUFT #MBßFOPWJˀ * ,JOE 5 +J + 'JFIO 0 4PěXBSF 5PPMT BOE "QQSPBDIFT GPS $PNQPVOE *EFO UJėDBUJPO PG -$.4.4 %BUB JO .FUBCPMPNJDT .FUBCPMJUFT


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Autonomous METLIN-Guided In-source Fragment Annotation for

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