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CSF and Tissue Samples: Human brain and serum/plasma samples were obtained from Maine Medical Center Research Institute BioBank (Scarborough, ME). Human CSF samples were obtained from Johns Hopkins School of Medicine, Alzheimer’s Disease Research Center (Baltimore, MD). Preparation of Protein Lysates and Digested Peptides: Tissue was pulverized under liquid nitrogen using a Bessman press, and transferred into Urea Lysis Buffer (ULB, 8 M Urea, 20 mM HEPES pH 8.0, 1 mM β-glycerophosphate, 1 mM sodium vanadate, 2.5 mM sodium pyrophosphate). The tissue slurry was homogenized using a mini-beadbeater and sonicated 3 times for 20 s each at 15 W output power with a 1 min cooling on ice between each burst. Lysates were centrifuged 15 min at 4 °C at 20,000× g to remove insoluble debris. CSF was added to dry aliquots of RapiGest (Waters) and ammonium bicarbonate (pH8.0) to a final concentration of 0.1% and 50 mM, respectively. Soluble protein was reduced with 4.5 mM DTT (30 min, 40 °C) and alkylated with 10 mM iodoacetamide (15 min, RT) in the dark. Samples were diluted 1:4 with 200 mM ammonium bicarbonate (pH 8.0) and digested overnight with trypsin-TPCK (1:75, w:w, Promega) in 1 mM HCl. Other protease digestions were performed using the manufacturer’s recommended protocol (LysC, AspN, GluC, ArgC, Promega and NEB). Digested peptide lysates were desalted over 360 mg SEP PAK Classic C18 columns (Waters, Richmond, VA, USA, #WAT051910). Peptides were eluted with 50% acetonitrile in 0.1% TFA, dried under lyophilization conditions, and stored at −80 °C in 0.1 – 1.0 mg aliquots. TAU and H3K9acetyl BAMS assays were performed using 100 µg of protein from human brain. Beta-amyloid BAMS assay was performed using 100 µL of undigested CSF. BAMS Assay - Bead Preparation: Each antibody was conjugated to NHS-activated magnetic agarose beads in a 10 µL slurry (7 µg of antibody/100 beads, 375 - 420 micron diameter) in PBS buffer, for 3-12 h (4 °C), and quenched with Tris HCl (100 mM, pH 8.0) for 1 h at RT. Unbound antibody was removed with three 400 µL washes of cold PBS (2 min, 4 °C). Beads were stored in PBS and 0.02% sodium azide (4 °C). BAMS Assay – Target Peptide Binding: Target peptide enrichment was performed using 10 – 1000 µg of digested peptides (or soluble protein) with typically 3 replicate beads/target. Multiplex peptide enrichment was performed using 10 – 1000 µg of purified peptides (or soluble protein) with typically 3 replicate beads/target in a volume of 50 – 200 µL. Peptides and affinity capture beads were incubated in binding buffer (1M KCl, 100 mM Tris HCl in deionized water, pH 8.0) for a period of between 3 – 12 hrs at 4 °C in Thermomixer (Eppendorf). Beads were washed sequentially in PBS (700 uL), ammonium bicarbonate (700 uL, 10 mM, pH 8.0) and deionized water (700 uL) to remove any nonspecific bound peptides (2 min, 4 °C). BAMS Assay – Target Peptide Elution: Washed BAMS beads are transferred to the hydrated wells to settle into the micro-wells of the BAMS plate assembly with gentle agitation and centrifugation (5 min, 200 x g). After centrifugation, the sample chamber gasket is removed, leaving the micro-well gasket fixed in place on the slide. The bead array is exposed to an aerosol of elution buffer using a Matrix Sprayer, containing 0.5 mg/mL α-cyano-4-hydroxycinnamic acid (CHCA) in 50% acetonitrile and 0.4% trifluoroacetic acid (TFA) for approximately 15 min. Once the matrix is dry, the silicone gasket is lifted off the slide and any remaining dry agarose beads are removed by compressed air leaving an array of spots containing purified and concentrated target peptides for subsequent MALDI MS measurement. MALDI TOF Linear Instrument Settings: Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) MS data was acquired on Bruker Daltonics (Billerica MA) Autoflex Speed or rapifleX MALDI-TOF/TOF mass spectrometer using FlexControl v.3.4 software. Unless otherwise indicated, the autoflex speed acquisition conditions in the positive linear mode were, 750-7000 m/z mass range (2 kHz, 10000 spectra/spot using the random walk method). The voltage settings were 19.50 kV (ion source 1), 18.35 kV (ion source 2) and 6.0 kV (lens). The pulsed ion extraction was 130 ns. The detector gain voltage was 4.0X or 2910 V. The acquisition settings on the rapifleX for automated runs in positive linear mode were 700-7000 m/z mass range, 10 kHz laser repetition rate, 4000 laser shots. MALDI TOF Analysis: Mass spectra were processed and analyzed using FlexAnalysis software. Peaks were detected that had a signal-to-noise ratio of at least 3. In some cases, baseline subtraction procedure was applied to individual mass spectra. Unless otherwise indicated, peaks in the mass spectra are labeled using either average or monoisotopic m/z values using the peak picking algorithms available in the software. mMass was utilized for data review.

INTRODUCTION METHODSProteomic studies critically rely on multi-dimensional separation methods such as 2D-LC to simplify the complexity of enzymatically digested biological specimens before subsequent MS & MS/MS to efficiently monitor multiple proteins of interest. The time and expertise required to implement LCMS methods can often be a barrier to using targeted proteomic applications in translational research. Here we present further development of a microarray analytical platform termed Bead Assisted Mass Spectrometry (BAMS), which integrates multiplex immuno-affinity capture with MALDI-TOF MS to create customized targeted proteomic assays for basic biology and neurobiology. We demonstrate efficient monitoring of multiple histone epigenetic modifications and several core Alzheimer’s disease pathological biomarkers including tau and amyloid beta peptides with a sensitivity greater than that of LCMS methods.

METHODS

METHODS&RESULTS

CONCLUSIONS• BAMS assays have been configured to enable multiplexed MALDI MS monitoring of both unmodified and phosphorylated sites within TAU in human brain & CSF samples. • A single on-bead, multiplex BAMS assay has been configured monitor up to 18 Beta-Amyloid fragments in CSF. • BAMS assays have also been configured for epigenetic applications to monitor Histone H3 K9acetylation containing peptides and associated combinatorial PTMs. • BAMS assays can be utilized to perform a wide range of targeted proteomic applications and is amenable for high-throughput screening.

1US patent 9,618,520 by inventor V.Bergo, titled Devices and methods for producing and analyzing microarrays 2US patent 10,101,336 by inventor V.Bergo, titled Eluting analytes from bead arrays 3US patent application 16/125164 by inventor V.Bergo, titled Multiplexed bead arrays for proteomics

ACKNOWLEDGEMENTS•  North Shore InnoVentures (https://nsiv.org/) and its corporate sponsors sand the

Massachusetts Life Science Center (http://www.masslifesciences.com/) for grant support.

Figure 1. BAMS Assay Workflow for Targeted Bottom-Up (or Middle-Down) Proteomics Applications. Standard bottom-up methods are used to generate proteolytic peptides for targeted monitoring of proteins using the BAMS workflow (lysis, reduction, alkylation, digestion). Digested peptides (C18 purified or directly from digestion reaction) are incubated overnight with BAMS affinity capture beads in Eppendorf tube or 96-well microtiter plate. For middle-down applications, such as CSF, beads can be incubated directly for peptide binding (1). Magnetic agarose beads are transferred, sequentially into wash buffers (PBS, ammonium bicarbonate, Milli-Q water) before placed into the BAMS slide (2). Washed beads are transferred into hydrated wells of BAMS chip with gentle agitation and a short centrifugation to settle beads into pico-wells (3), captured peptides from each bead are eluted into the pico-well using a matrix sprayer (4), eluted, dry peptides are analyzed after disassembling gaskets and placing into slide adapter for MALDI MS measurement (5).

RESULTS

JeffreyC.Silva1,SergeiDikler2,SergeyMamaev1,CamillaWorsfold1,AbhayMoghekar3,ThorstenWiederhold4,GhaithM.Hamza5,6,DonM.Wojchowski6,M.PaolaCastaldi5,ShirinArastuKapur7,VladislavB.Bergo11AdeptrixCorpora/on,BeverlyMA01915,2BrukerScien/fic,BillericaMA01821,3JohnsHopkinsUniversitySchoolofMedicine,Bal/moreMD21287,4CellSignalingTechnology,DanversMA01923,5AstraZeneca,BostonMA02451,6UniversityofNewHampshire,DurhamNH03824,7Cortexyme,SouthSanFranciscoCA94080

ATargetedMulEplexedMALDIMSAssayPlaIormusingAffinity-BeadAssistedMassSpectrometry(Affi-BAMS)forMonitoringBrainandCSFBiomarkers

Figure 2. Apparatus & Components for BAMS Assay. The BAMS assay components include: ITO or gold slides, pico-well gasket, sample chamber gaskets, clamps and centrifuge adapter (A). Antibody beads are provided separately. The matrix sprayer provides optimized elution conditions for MALDI MS measurement (B). Eluted peptides on ITO BAMS slides for low, medium and high-density assays (C). Slide adapter for BAMS slide and standard MALDI slide (D). Fluorescent labeled peptide on bead (E) and eluted peptide in pico-well (F). Jeffrey C. Silva | Adeptrix Corporation | 100 Cummings Center, Suite 438Q | Beverly, MA 01915 | jsilva@adeptrix.com

www.adeptrix.com

RESULTS

Figure 5. Subset of TAU BAMS Assays with LysC Digested Brain Sample. A) N-terminus of Tau (aa1-24) with loss of methionine and acetylation at the resulting n-terminal alanine, B) N-terminus of Tau (aa45-67), including signal from a phosphorylated site within the amino acid region 45-67, C) Internal TAU (aa88-130), D) Phosphorylated TAU (pT181) containing amino acids 175-190. E) Phosphorylated TAU containing amino acids 191-224 containing multiple sites of phosphorylation (pS198, pS199, pS202 & pT205). F) Phosphorylated TAU (pT231) with non-phosphorlyated and phosphorylated LysC peptide readout.

Figure 4. Regions of TAU Protein Configured for BAMS Assay. Panel A) TAU protein sequence (isoform Tau-F) with yellow highlighted areas indicating the corresponding LysC peptide sequences captured by BAMS assay using the antibody pictured below the corresponding region (rabbit monoclonal). Panel B) List of eight antibodies configured for BAMS assay to monitor specific regions of the TAU protein. Alternative proteases can be used to monitor the designated regions of the protein as defined by the specificity of the antibody. A list of the validated captured peptide sequences are shown for LysC, Trypsin and ArgC protease digestion conditions (or not detected, n.d.). Alternative digestions conditions can be used to monitor areas of the protein outside the epitope region if the resulting proteolytic peptide preserves the epitope sequence for efficient antibody binding.

Figure 6. TAU BAMS Assays with Normal (BLACK) and Disease (RED) Brain. A) Singly phosphorylated (pT231) and doubly phosphorylated (pT231 & pS235) TAU, Normalized to pT231, B) Phosphorylated TAU containing amino acids 191-224 containing multiple sites of phosphorylation (pS198, pS199, pS202 & pT205), normalized to singly phosphorylated pS199.

Figure 7. Beta-Amyloid BAMS Assay with Undigested Normal (RED) and Disease (BLACK) CSF. Overlaid MALDI MS spectrum from an on-bead multiplexing BAMS assay targeting the specific region of beta amyloid spanning from aa672-aa713 from triplicate bead assays of four patients (2 normal & 2 disease). The multiplex assay retrieves 19 specific beta-amyloid peptides between aa672-aa713. After normalizing all MALDI MS spectra to the Aβ1-40 peptide (BLUE BOX), clear separation of NORMAL and DISEASE patient samples is seen from specific beta-amyloid peptides (RED BOX).

Figure 8. Histone H3 K9acetyl BAMS Assay with Normal (BLACK) and Disease (RED) Brain. MALDI MS spectra obtained from replicate H3K9acetyl BAMS assay with equal quantities (100 µg) of LysC digested normal and disease brain (Panels A-E). Captured targets include H3 K9-acetylated peptides within aa1-23 of the H3 tail. Absence of mono-, di- and tri-methylated H3 K9acetyl peptides (2041, 2395 & 2625) are observed in diseased brain (Panels D & E). MALDI MS spectra obtained from H3K9acetyl BAMS assays with equal quantities (100 µg) ArgC digested normal and disease brain (Panels F-H). Captured targets include H3 K9-acetylated peptides within aa3-21 of the H3 tail, including combinatorial H3 tail peptides with acetylation, methylation and deimination of arginine (from conversion of arginine to citrulline), all containing lysine acetylation at K9 as directed by the site specific affinity capture of the BAMS assay. The MALDI MS shows four groups of ion clusters (1-4), with groups 1 & 2 (Panel G) related to ArgC fragments of K9-acetylated H3 (aa3-17) and groups 3 & 4 (Panel H) related to H3 tail clipping fragments (aa1-21) with ArgC missed cleavage due to arginine to citrulline conversion. Panel I shows duplicate normal and disease MALDI MS spectra normalized to a common H3 peptide at 1685.9 m/z, TK(me)QTARK(ac)STGGK(ac)APR.

Figure 3. BAMS Assay Validation Process. The specificity of affinity beads are validated by single bead affinity capture and localized peptide elution into the pico-well for direct, MALDI MS measurement of the peptide target in linear or reflector mode operation. Targeted MS/MS acquisition can be carried out further verify the identification of the captured peptide(s). A MALDI MS spectral library is generated for each protease digestion condition and each BAMS bead reagent (A). The BAMS assay can accommodate thousands of target peptides (unmodified & protein PTM) in a single experiment on a slide for identification and quantification of the target proteins in the configured assay panel (B).

magnetic beads

a) trypsin b) chymotrypsin c) others…

BA

A B D

E F

C

1.Enrich

2.Wash

3.AssembleBeadArray 4.EluteTargetPepEdes

5.ScanBAMSChip

microarray (bead array) MALDI scanner

AsliYleas10µgoftotalsoluble

protein

Overlay of 28-Plex BAMS Assay

Individual MALDI MS from Replicate BAMS assays

K.VAVVRtPPK.SMH+(Calc,mono)=1046.58

K.VAVVRtPPKsPSSAK.SMH+(Calc,mono)=1683.82

pT231

pT231&pS235

K.SGDRSGYssPGsPGtPGSRSRTPSLPTPPTREPK.KMH+(Calc,mono)=3533.68

1P

1P

K

80 80 80 2P 2P 3P

2P 3P

1955

.19

4329

.91

1827

.06

2068

.33

1561

.92

1196

.65

4131

.60

1698

.98

2314

.56

3262

.45

1325

.70

4230

.76

3787

.05

3673

.89

4513

.77

2167

.05

2461

.59

3133

.91

0

2

4

6

6x10

Inte

ns. [

a.u.

]

1000 1500 2000 2500 3000 3500 4000 4500m/z

MH+ = 1196.50, M.DAEFRHDSGY.E, aa672-681, Aβ1-10 MH+ = 1325.54, M.DAEFRHDSGYE.V, aa672-682, Aβ1-11 MH+ = 1561.67, M.DAEFRHDSGYEVH.H, aa672-684, Aβ1-13 MH+ = 1698.73, M.DAEFRHDSGYEVHH.Q, aa672-685, Aβ1-14 MH+ = 1826.78, M.DAEFRHDSGYEVHHQ.K, aa672-686, Aβ1-15 MH+ = 1954.88, M.DAEFRHDSGYEVHHQK.L, aa672-687, Aβ1-16 MH+ = 2067.96, M.DAEFRHDSGYEVHHQKL.V, aa672-688, Aβ1-17 MH+ = 2167.03, M.DAEFRHDSGYEVHHQKLV.F, aa672-689 Aβ1-18 MH+ = 2314.10, M.DAEFRHDSGYEVHHQKLVF.F, aa672-690, Aβ1-19 MH+ = 2461.17, M.DAEFRHDSGYEVHHQKLVFF.A, aa672-691, Aβ1-20 MH+ = 3133.44, M.DAEFRHDSGYEVHHQKLVFFAEDVGSN.K, aa672-698, Aβ1-27 MH+ = 3261.53, M.DAEFRHDSGYEVHHQKLVFFAEDVGSNK.G, aa672-699, Aβ1-28 MH+ = 3672.78, M.DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIG.L, aa672-704, Aβ1-33 MH+ = 3785.87, M.DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL.M, aa672-705, Aβ1-34 MH+ = 4073.00, M.DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG.G, aa672-708, Aβ1-37 MH+ = 4130.02, M.DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGG.V, aa672-709, Aβ1-38 MH+ = 4229.09, M.DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGV.V, aa672-710, Aβ1-39 MH+ = 4328.16, M.DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV.I, aa672-711, Aβ1-40 MH+ = 4512.28, M.DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA.T, aa672-713, Aβ1-42

Aβ1-10 Aβ1-11 Aβ1-13

Aβ1-16 Aβ1-28 Aβ1-33

Aβ1-34 Aβ1-37 Aβ1-38

Aβ1-39 Aβ1-40 Aβ1-42

2677

.175

1339

.904

Group-1, 2677.2, N-terminus (aa1-24) 806503/835203

0

1

2

3

4

5

5x10

Inte

ns. [

a.u.

]

1000 2000 3000 4000 5000 6000m/z

2390

.864

2470

.740

0.0

0.5

1.0

1.5

2.0

2.5

4x10

Inte

ns. [

a.u.

]

1000 2000 3000 4000 5000 6000m/z

4401

.923

2201

.549

3957

.010

0.00

0.25

0.50

0.75

1.00

1.25

5x10

Inte

ns. [

a.u.

]

1000 2000 3000 4000 5000 6000m/z

1153

.079

3453

.977

1791

.172

3582

.339

3662

.426

0

1

2

3

4

5

5x10

Inte

ns. [

a.u.

]

1000 2000 3000 4000 5000 6000m/z

3453

.977

3582

.339

3533

.937

3662

.426

K

79.96 80.09

0

1

2

3

4

5x10

Inte

ns. [

a.u.

]

3400 3450 3500 3550 3600 3650 3700m/z

1667

.641

0.0

0.5

1.0

1.5

2.0

2.5

4x10

Inte

ns. [

a.u.

]

1000 2000 3000 4000 5000 6000m/z

1683

.758

1047

.129

0

2

4

6

8

4x10

Inte

ns. [

a.u.

]

1000 2000 3000 4000 5000 6000m/z

1683

.758

1763

.542

1603

.777

79.98 79.78

0.0

0.2

0.4

0.6

0.8

1.0

5x10

Inte

ns. [

a.u.

]

1575 1600 1625 1650 1675 1700 1725 1750 1775 1800m/z

2390

.864

2470

.740

79.88

0

1

2

3

4x10

Inte

ns. [

a.u.

]

2360 2380 2400 2420 2440 2460 2480m/z

A B C

D E F

MH+ (calc) = 2766.26 -.MAEPRQEFEVMEDHAGTYGLGDRK.D

MH+ (calc) = 2391.03 K.ESPLQTPTEDGSEEPGSETSDAK.S

MH+ (calc) = 4401.09 K.QAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSK.S

MH+ (calc) = 1667.80 K.TPPAPKtPPSSGEPPK.S

MH+ (calc) = 3581.81 K. SGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKK.V

MH+ (calc) = 1683.82 K.VAVVRtPPKsPSSAK.S

1570

.604

2041

.173

2154

.355

1546

.591

1076

.262

2395

.521

1925

.061

2624

.163

0

1

2

3

4

5x10

Inte

ns. [

a.u.

]

1200 1400 1600 1800 2000 2200 2400 2600m/z

1076

.260

0.00

0.25

0.50

0.75

1.00

1.25

4x10

Inte

ns. [

a.u.

]

1060 1065 1070 1075 1080 1085 1090 1095m/z

1570

.609

0.0

0.5

1.0

1.5

2.0

2.5

5x10

Inte

ns. [

a.u.

]

1555 1560 1565 1570 1575 1580 1585 1590m/z

2041

.173

2154

.355

2055

.164

1925

.061

2069

.777

13.99 14.61

0.0

0.2

0.4

0.6

0.8

1.0

5x10

Inte

ns. [

a.u.

]

1925 1950 1975 2000 2025 2050 2075 2100 2125 2150m/z

2395

.533

2409

.604

2423

.945

2625

.288

2497

.669

2639

.281

2525

.376

2511

.240

2653

.156

14.07 14.34 14.1413.8813.99

13.57

0.0

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1.0

1.5

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4x10

Inte

ns. [

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2400 2450 2500 2550 2600 2650m/z

MH+ = 1076.2, K.QTARK(ac)STGGK.A, aa5-14 MH+ = 1546.8, -.ARTK(me)QTARK(ac)STGGK.A, aa1-14 MH+ = 1570.8, K.QTARK(ac)STGGK(ac)APRK.Q, aa5-18 MH+ = 1925.2, K.QTARK(ac)STGGK(ac)APRK(ac)QLA.T, aa5-21, TAIL CLIPPING MH+ = 2041.4, -.ARTK(me)QTARK(ac)STGGK(ac)APRK.Q, aa1-18 MH+ = 2154.5, K.QTARK(ac)STGGK(ac)APRK(ac)QLATK.A, aa5-23 MH+ = 2395.8, -.ARTK(me)QTARK(ac)STGGK(ac)APRK(ac)QLA.T, aa1-21, TAIL CLIPPING MH+ = 2625.1, , -.ARTK(me)QTARK(ac)STGGK(ac)APRK(ac)QLATK.A, aa1-23

B C

D E Mono-, Di- & Tri-Methyl

(See Panel D)

Mono-, Di- & Tri-Methyl (See Panel E)

A

2341

.220

2397

.605

2071

.272

2354

.962

2298

.662

2113

.428

2383

.445

1913

.344

2127

.439

2441

.227

2411

.002

1686

.048

2169

.474

2368

.746

1653

.746

2312

.277

1881

.217

2214

.455

2085

.276

1927

.189

1856

.740

1956

.348

2484

.355

1700

.199

2184

.230

1630

.164

2527

.791

2141

.496

1611

.828

2269

.989

1999

.437

2570

.700

2242

.770

0.0

0.2

0.4

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1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600m/z

2341

.220

2397

.605

2071

.272

2354

.962

2298

.662

2113

.428

2383

.445

2127

.439

2441

.227

2411

.002

2169

.474

2368

.746

2425

.539

2455

.857

2155

.462

2312

.277

2214

.455

2085

.276

2326

.006

2227

.885

2484

.355

2498

.741

2469

.417

2184

.230

2141

.496

2269

.989

2255

.792

2512

.122

2242

.770

14.09 14.15 14.27

18.2117.86

14.12

18.0117.88

13.84 14.81 14.34227.30

14.00 14.01 14.06 13.97 14.01 13.74 13.78

227.7942.16 42.56

0.0

0.5

1.0

1.5

2.0

2.55x10

Inte

ns. [

a.u.

]

2100 2150 2200 2250 2300 2350 2400 2450 2500m/z

1913

.344

1899

.226

1686

.048

1671

.957

1653

.746

1881

.217

1927

.189

1856

.740

1942

.003

1956

.348

1839

.193

1700

.199

1999

.437

1714

.471

1970

.283

1984

.401

14.00 14.01 14.06 13.97 14.01 13.74 13.78

227.7942.16 42.56

17.8618.21

14.09 14.15 14.27

17.8818.01

14.12 13.84 14.81 14.34227.30

0.00

0.25

0.50

0.75

1.00

1.25

1.50

5x10

Inte

ns. [

a.u.

]

1650 1700 1750 1800 1850 1900 1950 2000m/z

MH+ = 1685.9, R.TK(me)QTARK(ac)STGGK(ac)APR.K

MH+ = 1913.2, -.ARTK(me)QTARK(ac)STGGK(ac)APR.K

MH+ = 2071.4, R.TKQTARK(ac)STGGKAPR(citr)KQLA.T

MH+ = 2298.7, -.ARTKQTARK(ac)STGGKAPR(citr)KQLA.T

+ AR (Δ228)

+ AR (Δ228)

❶&❷

❸&❹

❶❷ ❸❹ F G

H

I

A

B