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Q U I C K S TA R T ( c o n t . )

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Cell Culture, Serum & Tissue: MKN-45 and HeLa cells were prepared in RPMI media with 10% fetal bovine serum (FBS) and 1X Pen-Strep (Sigma, #P4333) to 75% confluence at 37 °C with 5% CO2. Prior to chemical treatment, cells were serum starved in RPMI media with 0.2% FBS and 1% Pen/Strep for 12 hrs. SU11274 (SU) and staurosporine (ST) were used at a final concentration of 1 μM and 0.2 μM, respectively in 0.05% DMSO. Hydrogen peroxide (H2O2) was used at a final concentration of 2 mM with a 30 m pre-treatment of 0.1 mM sodium orthovanadate. Rapamycin treatment was carried out for a duration of 2 hours at a concentration of 1 mM. Serum and samples were obtained from commercial sources. Human brain samples were obtained from the Maine Medical Research Institute Biobank repository.

Preparation of Protein Lysates and Digested Peptides: Cells were washed twice with cold PBS. PBS was removed and cells were scraped in Urea Lysis Buffer (ULB, 8 M sequanal grade Urea, 20 mM HEPES pH 8.0, 1 mM β-glycerophosphate, 1 mM sodium vanadate, 2.5 mM sodium pyrophosphate). Tissue was pulverized under liquid nitrogen using a Bessman press, and transferred into ULB. The tissue slurry was homogenized using a mini-beadbeater. Cell cultures material and homogenized tissue were sonicated 3 times for 20 s each at 15 W output power with a 1-minute cooling on ice between each burst. Sonicated lysates were centrifuged 15 min at 4 °C at 20,000× g. An aliquot of each supernatant was reserved for Western blotting (if needed) and stored at −80 °C. Supernatants were collected and reduced with 4.5 mM DTT for 30 min at 40 °C. Reduced lysates were alkylated with 10mM iodoacetamide for 15 min at room temperature in the dark. Samples were diluted 1:4 with 0.2% 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.

Immunoaffinity Enrichment & MALDI Analysis: Protein A/G beads were prepared using NHS-activated XL magnetic agarose beads (400 micron, Cube Biotech) with Protein A/G (Abcam, 1 mg/ml) in PBS buffer. Antibodies (2 µg) were conjugated to 5 µL slurry of Protein A/G beads by overnight incubation in PBS with 0.1% BSA. Unbound antibody was removed with three 400 µL washes of PBS with 0.1% BSA. Individual target peptide enrichment was performed using 40 – 1000 µg of purified peptides with 1 – 3 beads. Multiplex target peptide enrichment was performed using 40 – 1000 µg of purified peptides with 3 beads per protein target. Peptides were incubated overnight at 4 °C. Beads were washed three times in PBS to remove nonspecific bound peptides. Stringent wash conditions included PBS with 0.5 – 1.0 M sodium chloride. Final washes included 10 mM ammonium bicarbonate (pH 7.5) and distilled water (MilliQ). Washed beads were transferred to ITO (or gold) slides affixed with the pico-well gasket and a sample gasket. Pico-wells were hydrated prior to adding washed beads using a benchtop swinging bucket centrifuge at low speed (~ 1000 xg). For single bead analysis, all liquid was removed from last wash and add 1.5 – 2.0 µL of matrix (10 mg/mL CHCA in 50% ethanol/water, 0.1% formic acid) to elute bound peptide(s) for 15 min at 25 °C. Spot 1uL of eluted peptides (in matrix) onto the MALDI plate. Allow to dry completely before MS analysis using a MALDI TOF instrument (Autoflex Speed, Bruker & SimulTOF ONE, SimulTOF).

INTRODUCTION   METHODS  Proteomic studies that monitor protein and PTM abundance often employ multi-dimensional analytical methods such as nano-LC-ESI-MS/MS to simplify the inherent sample complexity and wide dynamic range of endogenous proteins within biological specimens. The time and expertise required to implement and LCMS workflow can often be a barrier to integrating targeted proteomic applications for a particular translational research program. In addition, sample quantity requirements limit accessibility of LCMS-based targeted methods as a practical screening platform. In this study, we present a versatile microarray assay platform (BAMSTM) that integrates immuno-affinity capture with MALDI MS detection, which can be leveraged to perform both in vivo targeted proteomic assays as well as in vitro enzyme-substrate assays for a wide range of high-throughput screening applications.

METHODS  

METHODS  &  RESULTS  

CONCLUSIONS  •  Multiplexing of BAMS enables one to monitor 100’s of proteins in a single assay. •  The versatility of the BAMS allows rapid configuration of targeted assays

monitoring many aspects of the protein (unmodified, phospho-, acetyl- &, methyl-). •  BAMS assays are an efficient method to monitor a wide variety of proteins in any

type of biological samples. •  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 sponsor sand the Massachusetts

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

Figure 1. Sample Preparation Workflow. Standard bottom-up methods are used to generate proteolytic peptides for subsequent BAMS analysis (lysis, reduction, alkylation, digestion). Purified peptides are incubated overnight with BAMS affinity capture beads in eppindorf tube or 96-well microtiter plate (1). Magnetic agarose beads are transferred, sequentially into wash buffers (PBS, ammonium bicarbonate, DDW) 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  

Sergey  Mamaev  ,  Jeffrey  C.  Silva,  Camilla  Worsfold  &  Vladislav  B.  Bergo  Adeptrix  Corpora/on,  Beverly,  MA  01915  

A  High-­‐Throughput  MulJplexed  Assay  PlaNorm  for  Monitoring  Protein  Abundance  in  96-­‐Well  Cell  Cultures  or  Product  Profiles  from  Enzyme-­‐Substrate  ReacJons  

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) & 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. Forward and Reverse Curves for the PCI Peptide in Normal Human Serum using BAMS. MALDI MS peptide signal from BAMS assay of PCI & sTfR in normal human serum (reflector mode). The endogenous concentration of the PCI peptide is determined by the plateau shown by the forward dilution curve (FWD) and the analytical sensitivity of the assay is revealed by the reverse dilution curve (REV) when conducted using SIS standards in triplicate. The average %CV for the analytical replicates was deteremimed to be 15% and the dynamic range for the PCI peptide spanned approximately 3 orders of magnitude.

Figure 4. BAMS assay from Petri Dish and 96-Well Plate Cultures. MALDI MS signal from single bead peptide elution onto a BAMS assay slide for 4EBP1 (total), 4EBP1 (T37 & T46), AKT (total), A - F. Petri dish samples (A - C) were harvested from a SILAC experiment (light = control, heavy = peroxide, 250 µg total peptides), and BAMS assays were performed with medium density pico-well gasket (500 µm diameter wells, 400 µm beads). BAMS assays were conducted using peptides from 96-well cell culture (D - F) were performed with high density pico-well gasket (250 µm diameter wells, 200 µm beads, 10 µg total peptides).

Figure 6. BAMS assay to Monitor PTM Status. (Unmodified & Phosphorylated proteins as well as WT & MT isoforms). MALDI MS signal from a BAMS assay for unmodified and phosphorylated (S101) 4EBP1 (A) as well as singly and doubly phosphorylated 4EBP1 (T37 & T46) with 1 (BLUE) and 2 (RED) missed cleavages (B) from peroxide treated MKN45 cells. Wild-type and point mutant (A107V) for 4EBP1 from HCT116 cells (C & D).

Figure 7. BAMS assay to Monitor Protein Acetylation & Isoforms. Reflector mode MALDI MS signal from BAMS assays for VDAC1 & VDAC2. The captured N-terminal peptides to VDAC1 (A) and VDAC2 (B) were generated from LysC digested cells (8 cell line mix). The VDAC1 peptide, (M) A(Acetyl)VPPTYADLGK (S) (amino acids 2-12, calc. MH+ = 1173.615), and the VDAC2 peptide, (M) A(Acetyl)THGQTCARPMCIPPSYADLGK (S) (amino acids 2-23, calc. MH+ = 2473.142), are both N-terminally processed with loss of methionine and addition of N-terminal acetylation.

Figure 9. Monitor Multiple Sites of a Single Protein using BAMS. Multiple sites within a single protein can be monitored in a multiplex BAMS assay using validated antibodies to different regions of the target protein. An example is shown using three different affinity capture beads to 4EBP1, with the typical tryptic peptides highlighted in yellow and orange. Targeted peptide regions can be adjusted by using a different protease as illustrated above for LysC.

Figure 8. BAMS assay to Monitor Multiplexed Ubiquitination Reactions. Black trace -MALDI signal from a BSA digest spiked with 6 K-ε-GG containing peptides at 1.25µM each. None of the peptides were identified. Red trace – MALDI signal from BSA / K-ε-GG containing peptides after incubation with anti- K-ε-GG BAMS beads for 2 hours, washing with ammonium bicarbonate and eluting captured peptides from K-ε-GG affinity beads onto MALDI slide. Five out of six K-ε-GG peptides were identified after BAMS assay (green box).

Figure 3. BAMS Assay Validation Workflow. The specificity of affinity beads are validated by single bead affinity capture and localized peptide elution into the pico-well for MALDI MS and/or MS/MS to identify captured peptide(s). A MALDI MS spectral library is generated for each protease digestion condition and each Affi-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…

B  A  

A B D

E F

C

~15 fmoles

~ 3 Orders of Magnitude Dynamic Range

PCI peptide: EDQYHYLLDR

sTfR, GFVEPDHYVVGAQR

PCI (PAI3), EDQYHYLLDR

M. Razavi et al, Clin Chem (2013) 59(10): 1514-1522.

UNMODIFIED & PHOSPHORYLATED WILD-TYPE

POINT MUTANT (A107V) SINGLE & DOUBLE PHOSPHORYLATION

RAGGESSQFEMDI

RAGGESSQFEMDI RVGGESSQFEMDI

RNSPEDKRAGGESSQFEMDI

Δ80

Δ28

Δ80 Δ80

❶ ❷ ❶

❷

A

B RVVLGDGVQLPPGDYSTTPGGTLFSTTPGGTR

C

D

RVVLGDGVQLPPGDYSTTPGGTLFSTTPGGTR FLMECRNSPVTKTPPR RAGGEESQFEMDI

1.  Enrich  

2.  Wash  

3.  Assemble  Bead  Array   4.  Elute  Target  PepJdes  

5.  Scan  BAMS  Chip  

microarray (bead array) MALDI scanner

As  li[le  as  10  µg  of  total  soluble  

protein  

Overlay of 28-Plex BAMS Assay

Individual MALDI MS from Replicate BAMS assays

Petri Dish (10cm) 96-Well Plate

RAGGESSQFEMDI MH+ = 1468.637, Δ10

NSPEDKRAGGESSQFEMDI MH+ = 2138.929, Δ18

Δ10 Δ20

Δ10

RPHFPQFSYSASGTA MH+ = 1652.782, Δ10

VVLGDGVQLPPGDYSTTPGGTLFSTTPGGTR, MH+ = 3207.465 RVVLGDGVQLPPGDYSTTPGGTLFSTTPGGTR, MH+ = 3363.566

A

B

C

D

E

F

Ave FC = -1.1

Ave FC = 1.6

Ave FC = 1.0

J. E. Rodriguez et al, Curr Hypertens Rep (2009) 11(6): 396-405.

K-ε-GG Enrichment

& MALDI MS

Trypsin Digestion E2 E1 E3

in vitro substrate proteins

Ubiquitin Ligase

Ubiquitinated substrates

A B VDAC1: A(Acetyl)VPPTYADLGK VDAC2: A(Acetyl)THGQTCARPMCIPPSYADLGK