One-Pot Isotope Dilution PRM for Quantitative Protein Heterogeneity in Red Blood Cells
2.4 Modified Rapid Digestion Trypsin/Ly-C
The protocol used for sample preparation was based on previous studies 10,17–19 but was modified to eliminate surfactant, alkylating agent, solid-phase extraction and automated low-volume liquid handling instrumentation. Microlitre volume, one-pot, organic solvent assisted, solid phase extraction-free sample preparation was performed as follows: Unless otherwise noted, all reagents were LC-MS grade. To wells containing RBC(s), encapsulated in HSA, 10 µl of 10% MeCN in water was added. The OP384 was covered with the accompanying lid and placed in a bath ultrasonic cleaner (Digital Ultrasonic Cleaner, PS-40A, Vevor, USA) operated at 240 W and 40 KHz with bath water temperature of 70˚C for 15 min. The bath sonicator was covered with a lid to ensure 100% humidity and Styrofoam around the perimeter of the OP384 assisted level floatation in the bath (preventing flooding of the wells with bath water). After the 15 min incubation the OP384 was placed in an ice bath to minimize sample evaporation. A modified rapid digestion protocol (Rapid Digestion Kit-Trypsin/Lys-C, CAT.#VA1061, Promega, USA) was used substituting the rapid digestion buffer with 25 mM Tris pH 8, 2 mM Bond-Breaker TCEP solution (Thermo Scientific, USA, CAT.#77720). The Trypsin/Lys-C mix was diluted to 100 ng/µl in water and mixed with the substitute digestion buffer at a 1:15 (v/v) and 16 µl of enzyme in digestion buffer was added per well (in the OP384). Iodoacetamide was not included as an alkylating agent based on a previous study which demonstrated increased N-terminus peptide alkylation at temperatures exceeding 40˚C 231. While not performed in this study, alkylation with 40 mM iodoacetamide for 30 min at room temperature has been tested and may be included, after high temperature digestion to prevent inter-peptide cysteine-cysteine bonds. High temperature digestion was achieved by placing the OP384 in the bath sonicator as described above but without sonication for a 60 min incubation followed by 20 min of sonication at 240 W and 40 KHz. The OP384 was then placed in an ice bath and acidified with 1 µl of 10% formic acid and spiked with 2 µl of stable isotope labeled peptide standards (SIL) at a concentration of 1 fmol/µl. As silicone mats are not commercially available for the OP384, aluminum foil was used instead and fixed in place using labeling tape at which point samples were considered prepared for nano-LC-MS. The volume of sample injected per LC-MS analysis was 15 µl which was approximated to be equivalent to ~5 µg of protein, based on ~80 nl of sample (containing 6.67% HSA) spotted per well.
2.5 Nano-LC-MS/MS at site 1
Nano-LC-MS at site 1 (University of Victoria) encompassed PRM assay development, including LOD, repeatability and specimen analysis. Nano-LC-MS was performed using EASY-nLC 1000 Liquid Chromatography instrument (Thermo Scientific) and an Orbitrap Fusion ETD Tribrid Mass Spectrometer (Thermo Scientific) operated using Xcalibur software version 4.3.73.11 (Thermo Scientific).
The EASY-nLC 1000 was operated in the two-column mode using in-house packed (MAGIC C18AQ 100A 5 µm, Michrom Bioresources, USA) trap column (2 cm, 75 µm ID) upstream of an in-house packed (MAGIC C18AQ 100A 5 µm, Michrom Bioresources) analytical column (15 cm, 75 µm ID). While the 5 µm beads do not offer the same separation resolution as 3 µm and 2 µm beads, experience (in-house) has demonstrated them more robust and compatible with targeted quantification for trace materials, including single cell input. It requires noting, the MAGIC C18AQ 100A 5 µm from Michrom Bioresources may no longer be commercially available and the Princetonpher-100 C18 100-5U (Princeton Chromatography, USA) may serve as a potential substitute. Flow rate was maintained at 300 nl/min resulting in a back pressure range of ~100-140 bar throughout the gradient, at ambient column temperature (22-27˚C). Mobile phase A and B consisted of 0.1% formic acid in water and 0.1% formic acid in 90% MeCN, respectively. The elution gradient was performed as follows; 0% B 0 min, 40% B 20 min, 80% B 2 min, 100% B 4 min, 0% B 6 min, total gradient time of 32 min. Pre-column and analytical columns were equilibrated with 4 µl each mobile phase A at variable flow rates determined by setting an instrument controlled maximum pressure of 348 bar. Sample loading onto the trap-column was performed at a flow rate of 4 µl/min with maximum pressure set at 348 bar. The total time for equilibration and sample loading was ~15 min and the total time for analysis was 47 min per sample. A needle wash sequence consisting of five 25 µl volumes each of polar and organic solvents was programmed to be performed during the gradient to minimize cross-sample carryover between injections. Electrospray was achieved by union of the analytical column with an emitter consisting of a 10 µm tip (PicoTip Emitter, New Objective, USA, FS360-20-10-N-20-C12). Note, since solid phase extraction is omitted a precolumn filter of 0.2 µm porosity fitted directly to the switching valve and upstream of the line-out should may be considered to prevent particulates from entering the flow path which otherwise may increase system pressure and/or clog downstream components. Additionally, the well coordinates for injecting from an OP384 plate are not pre-programmed in the EASY-nLC 1000 software and required calibration using the instructions provided in the user guide (Thermo EASY-nLC 1000 User Guide (Touch-screen software version 3.0), 60053-97227 Revision C January 2012).
The nano-LC method for data dependent acquisition (DDA) and data independent acquisition (DIA) was identical to that described above for PRM analysis apart from the elution gradient which was as follows: 0% B 0 min, 25% B 100 min, 40% B 20 min, 90% B 15 min, 100% B 1 min, 0% B 4 min for a total gradient time of 140 min.
The Xcaliber acquisition settings for collecting MS spectra were based on previously developed methods for PRM (PRM_FusionLumos_SW3_1.pdf (washington.edu)), DDA and DIA 24 modes and the details of the settings used in the present study are provided in supplemental Figures S1, S2 and S3-S4, respectively.
2.6 Nano-LC-MS/MS at site 2
Nano-LC-MS at site 2 (Brigham Young University) utilized PRM parameters developed at site 1 to reproduce LOD, perform LOQ and measure endogenous targets in RBCs prepared by automated preparation in one-pot for trace samples (AutoPOTS) 18. The LOD and LOQ were determined using a seven-point calibration curve consisting of the following peptide standard concentrations (in amol/µl): Blank 0, 0.975, 3.9, 15.6, 62.5, 250 and 1000, the SIL peptides were used as normalizers spiked-in at constant concentration of 200 amol/µl. Triplicate injections were performed at each concentration to evaluate the intra-assay CV and to define the LOQ for use as Tier 2 assays for research application 25. A similar but not identical (to site 1) nano-LC method was used consisting of online SPE and a two column setup; precolumn (Jupiter 3.0, C18, 100 µm ID, 5 cm) (Phenomenex, Torrance, CA) and an analytical column (Dr. Maisch 3.0 µm, 30 µm ID, 50 cm). The LC gradient was 2% to 40% B (20 min); 40% to 80% (2 min) and 80% to 99% (4 min) at a flow rate of 0.535 µL/min (1/9 split was applied before the analytical column). The LC and MS instruments at site 2 were an UltiMate 3000 RSLCnano with a UltiMate™ WPS-3000TPL/PL RSLCnano well Plate Autosampler (Thermo Fisher, Waltham, MA) and an Orbitrap Exploris 480 (Thermo Fisher) mass spectrometer with the Nanospray Flex ion source.
Endogenous peptide measurements in RBCs isolated using FACS was performed as previously described 18. One microliter of mobile phase A (water with 0.1% FA) was dispensed into each well prior to FACS sorting and RBCs were deposited in four replicates at concentrations of 5, 50 and 250 RBCs per well into Coring low volume 384-well plate (PN 3544). Two microliters of 0.5% N-Dodecyl-β-D-maltoside (DDM) in 50 mM Ammonium bicarbonate (ABC)/phosphate buffered saline (PBS) was added into each well for cell lysis and protein denaturation for 60 min at 70 ⁰C. One microliter of enzyme solution with 2 ng Lys-C in 50 mM ABC was added and incubated at 37 °C for 4 h. Followed by a 12-hour 37 °C trypsin digestion step, with 1 µl of enzyme solution containing 2 ng trypsin in 50mM ABC. One microliter of 5% formic acid solution was used to quench the digestion, and 2 µl of 800 amol SIL peptide standards (VHLTPEEK(+8) and CM-VHLTPEEK (+8)) was spiked in. The 384 well plate was then sealed with a foil adhesive mat and stored at −20 °C. Nano-LC-MS was performed by injecting 7 µl of each sample using the same column and gradient setup described above for LOD and LOQ experiments. Samples were injected in an ascending sequence from low to high concentrations. PRM was performed using the method developed at the University of Victoria but on a Orbitrap Exploris instrument (Thermo).
2.7 Peptide Standard Synthesis
Fmoc solid phase synthesis of the peptide VHLTPEEK, which can exist on the N-terminus of human hemoglobin beta subunit (HBB_HUMAN, UniProtKB - P68871) after N-terminal methionine excision, and of the advanced glyucation product carboxymethyl valine (CM- VHLTPEEK) were performed as previously described 26,27, with modification. Natural (NAT) and stable isotope labeled (SIL) versions of the peptide were synthesized by incorporating 12C/14N amino acids for the NAT peptide and 13C/15N heavy isotope labeled L-lysine on the C-terminus of the SIL peptide. Peptides were synthesized in duplicate and prior to deprotection one pair of NAT and SIL peptide were subjected to on-resin N-terminal carboxymethylation. Carboxymethylation was achieved using glyoxylic acid and sodium cyanoborohydride treatment as previously described 27 with modification. In a 5 ml glass vial, 21 mg of HCOCO2H · H2O (Sigma Aldrich, Canada, G10601) and 47 mg NaBH3CN (Sigma Aldrich, Canada, 156159) were dissolved in 5 ml of dimethyl formamide (DMF). From this solution 200 µl was added to peptides still protected and coupled to resin and incubated for 12 h at ambient temperature in a fume hood. The reaction solution was subsequently eluted, and 3 replicate washes were performed using 200 µl DMF. The other pair of NAT and SIL peptides were treated to a similar protocol omitting addition of HCOCO2H · H2O and NaBH3CN. Peptides were subsequently deprotected and decupled from the solid phase resin using repeated incubations with a solution of trifluoroacetic acid, triisopropylsilane and water (23.75:0.625:0.625 ratio, respectively). The eluents were precipitate with cold ether, evaporated and subsequently freeze dried. Peptides were re-solubilized in 100 µl of 3% MeCN and subjected to matrix assisted laser desorption ionization time of flight MS analysis using an Ultraflex III-MALDI TOF/TOF Mass Spectrometer (Bruker LTD., Milton, ON) for crude yield and purity assessment.
Peptides were purified by preparative HPLC using an Agilent 1290 Infinity II UHPLC equipped with an Eclipse Plus C18 RRHD 1.8 µm 2.1 x 150 mm analytical column (Agilent). Retention times for peptides were previously determined on the same LC and column using dynamic MRM on an Agilent 6495B Triple Quadrupole MS. Purity analysis for each fraction was performed using an Ultraflex III-MALDI TOF/TOF Mass Spectrometer (Bruker LTD., Milton, ON) and only pure fractions were pooled for subsequent amino acid analysis. The purity of the pooled fractions was confirmed via UV-HPLC on an Agilent 1290 Infinity HPLC instrument.
Amino acid analysis was performed as previously described 28 with modification. Peptide standards were subjected to acid hydrolysis and subsequent derivatization via dansylation. Quantification via dynamic MRM was performed on an Agilent 1290 Infinity II UHPLC online with a 6495B triple quadrupole MS using 13C or 2H labeled internal standard for each amino acid (Cambridge isotope Laboratories, Inc., MA, USA), except for the carboxymethyl valine for which a standard was not available.
2.8 Carboxymethyl Valine Stability at High Temperature and Multiple Reaction Monitoring
The stability of the carboxymethyl modified peptide standard (CM-VHLTPEEK) and the unmodified standard (VHLTPEEK) were compared at 70˚C and 4˚C as follows. A working stock of SIL and NAT peptides was made to consist of ~50 fmol/µl of each peptide in 3% MeCN and 3 ul was added in triplicate to 5 µul of 10% MeCN pre-dispensed in 3 separate wells of a Greiner low volume 384 well microplate (8 µul final in each well). The exact triplicate composition and volume was prepared in 300-μl sample injection vials (PP screw vial, 12 × 32 mm, 9 mm thread, from Canadian Life Science, Edmonton, AB). The microplate was placed in a sonicator bath at 70 ˚C and sonicated for 15 min while the vial samples were stored in a refrigerator (4 ˚C). After the incubation, the microplate was placed on an ice bath and allowed to cool. To the microplate and sample injection vials 16 µl of 25 mM Tris pH 8, 2 mM TCEP was added followed by 1 µul of enzyme (Promega rapid digest Ttrypsin/LysC). The microplate was incubated at 70 ˚C for 1 h followed by 20 min sonication, the sample injection vials were incubated in the refrigerator (4 ˚C). To each well, 1 µl of 10% formic acid was added and samples were analyzed via dynamic multiple reaction monitoring (MRM) using an Agilent 1290 Infinity II LC System and Agilent 6495B Triple Quadrupole MS instruments (Agilent) as previously described 29,30, with modification. Five microliters of sample was injected and separation was achieved using an Eclipse Plus C18 RRHD 1.8 µm 2.1 x 50 mm analytical column (Agilent) fitted with an upstream 1290 Infinity II in-line filter, 0.3 µm, 2 mm ID, SST (Agilent). The gradient was as follows; 2% (2 min); 2% to 25% (5 min) at a flow rate of 0.4 ml/min, 25% to 98% (2 min) at a flow rate of 0.6 ml/min and 98% to 80% (2 min) at a flow rate of 0.4 ml/min followed by a post time of 2 min of 2% B at a flow rate of 0.4 ml/min. The aqueous and organic mobile phase buffers consisted of 0.1% formic acid in water and 0.1% formic acid in MeCN, respectively. For the first 2 min of the gradient the flow from the LC was diverted to waste, between minutes 2 and 7 the flow was diverted to the mass spectrometer and between minutes 7 and 11 the flow was diverted to waste. Targeted MS acquisitions were performed at unit resolution using 6-min detection windows, a 890-ms cycle time, resulting in a dwell time of ~10 ms. Three time segments were used for dynamic MRM as follows: at a start time of 0 min, the valve was diverted to waste, at 2 min the valve was diverted to the MS, and at 7 min the valve was diverted to waste and the Delta EMV (+) was set to 500. The source settings were set as follows; gas temperature 150 °C, gas flow 15 L/min, nebulizer 30 psi, sheath gas temperature 250 °C, sheath gas flow 11 L/min, capillary 3500 V and 3000V (-ve) for positive and negative ion funneling, respectively, nozzle voltage was 300 V and 1500 V for positive and negative ionization, respectively, iFunnel parameters for high pressure RF was 200 V for positive mode and 150 V for Negative mode and low pressure RF was 110 V and 60 V for positive and negative, respectively.
2.9 Analysis of Blood Specimens from Study Participants
Endogenous CM-VHLTPEEK and VHLTPEEK in blood samples were quantified via low cell input one-pot PRM and by standard bulk sample preparation and MRM. Detailed chronological specimen collection and processing for low cell input one-pot PRM is provided in supplemental_document_1. After spotting, samples were processed as described in 2.4 and analyzed as described in section 2.5. In addition to the specimens analyzed a background control (HSA without RBC) was also subjected to 10 replicate spotting and processed in parallel as described in sections 2.4 and 2.5. Lastly, variability of the analytical workflow was assessed by preparing CM-VHLTPEEK and VHLTPEEK peptide standards in mobile phase A with identical SIL concentrations (to that used for specimens) and NAT concentrations at average endogenous (in RBCs) CM-VHLTPEEK and VHLTPEEK concentrations, respectively. Briefly, a master mix was made in a background of 0.1% FA in water. Peptide standards were spiked int at concentrations so that when 1 µl of the mix was injected for PRM the following amounts of peptide standards would be injected on column: 13 amol CMV-NAT, 12.7 fmol NAT, and 2 fmol each CMV-SIS and SIS. As with specimens and HSA control, 10 injections were analyzed as described in section 2.5.
Bulk sample preparation used 50 µl of gravity-packed RBCs dispensed into 1 ml lysis/protein solubilization buffer consisting of 2% sodium deoxycholate and 50 mM ammonium bicarbonate in water. Cell disruption was achieved by incubating at 99 ˚C on a Thermo Mixer (Eppendorf, Hamburg, Germany) at 1000 rpm shaking for 15 minutes. Proteins were reduced and alkylated with 20 mM DTT and 40 mM IAA and 1:20 and 1:50 dilutions used for protein quantification using the Bradford assay. Trypsin digestion was achieved using a 1:1 trypsin to protein concentration ratio and incubating at 37 ˚C for 9 h with 750 rpm shaking on a Thermo Mixer (Eppendorf). Acidification to a final concentration of 1% formic acid was used to stop the digestion and precipitate the sodium deoxycholate. Samples were subjected to solid phase extraction, lyophilization, and subsequent solubilization in 0.1% formic acid to a protein concentration of 0.6 µg/µul. A 25 µl aliquot of the sample was mixed with 25 µul of SIL and CMV-SIL at 8000 fmol/µl and 80 fmol/µl, respectively. Using 5 µl, samples were analyzed via MRM as described in section 2.8. Endogenous peptides were quantified using external calibration curves. Nine-point calibration curves were generated in mobile phase A using SIL and CMV-SIL peptides as normalizers, at identical concentrations to those used for specimens. The NAT and CMV-NAT peptide concentrations reflected expected endogenous concentrations with ranges of 16 fmol/µl to 107 513 fmol/µl and 0.18 fmol/µl to 1200 fmol/µl, respectively.
2.10 Selectivity
Selectivity for measuring endogenous CM-VHLTPEEK was assessed as previously described 31. The calibration curves from the bulk analysis, described in 2.9 revealed an LOQ of 75 fmol CM-VHLTPEEK injected on column. Based on this LOQ, three-point calibration curves were generated in N=6 bulk sample preparations, described in 2.9, with the lowest point on the calibration curve being endogenous signal, the middle point was spiked in at 25X the LOQ (75 fmol X 25 = 1875 fmol NAT CM-VHLTPEEK injected on column) and the highest point on the curve was 50X the LOQ (75 fmol X 50 = 3750 fmol CM-VHLTPEEK on column). SIL CM-VHLTPEEK peptide was spiked-in at 30 fmol/µl (600 fmol on column). Samples were prepared in duplicate and each sample was injected in replicate for MRM analysis (described in 2.8) with a wash injection included between each injection. Samples were prepared to a 60 µl final volume in each sample injection vial as follows: 55 µl trypsin digested sample, described in 2.9, and 5 µl of peptide standard. The high concentration peptide standard mix contained 360 fmol/µl CMV-SIL and 2250 fmol/µl CMV-NAT. The medium concentration contained 360 fmol/µl CMV-SIL and 1126 fmol/µl CMV-NAT. And the lowest concentration contained 360 fmol/ul CMV-SIL. Twenty microliter injections were used for MRM analysis which was performed as described in 2.8.
2.11 Data Analysis and Availability
Datasets for targeted experiments (PRM and MRM) are available through Panorama Public 32 which can be searched using the title of this manuscript. Datasets for DDA and DIA have been deposited and are available from the Center for Computational Mass Spectrometry using the MassIVE identifiers MSV000088397 and MSV000088395, respectively.
Data acquired using DDA and DIA were analyzed using the MSFragger 33 and DIA-Umpire 34, the default workflow was selected in FragPipe version 15.0 with MSFragger version 3.2 and Philosopher version 3.4.13 (build 1611589727). Detailed parameter settings and search log files are provided as text files in the supplemental materials.
All targeted datasets were analyzed using Skyline 35. For associated figures generated in Graphpad Prism (GraphPad Prism version 8.2.1 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com), Skyline calculated peak areas were exported to Microsoft Excel (Microsoft Corporation, 2018. Microsoft Excel, Available at: https://office.microsoft.com/excel.) to perform calculations that were not available directly through Skyline, such as percent difference of the slope of the line across response curves in replicate biological matrix, cumulative percentile plots for analyzing repeated measures in blood specimens and other similar data analyses.
For LOQ and repeatability determination, a coefficient of variation of less than 20% across triplicate injections was set as the cut-off and the LOD was based on the Skyline calculated value. This data analysis pertains to data generated as described in 2.6.
Raw data files obtained from repeated measures of 5-25 RBCs in patient blood samples, control HSA samples and peptide standards spiked-in mobile phase A were analyzed as described above. Normalized peak areas for endogenous CM-VHLTPEEK and VHLTPEEK were used to calculate % CM-VHLTPEEK which were then plotted in Graphpad Prism using cumulative frequency distribution, selecting relative frequency, auto for center of first bin and center of last bin and selecting “No bins. Tabulate exact cumulative frequency distribution”. For normality and lognormality tests, normal (Gaussian) distribution, lognormal distribution and compute the relative likelihood of sampling from Gaussian (normal) vs a lognormal distribution (assuming no other possibilities) was selected. The method to test the distribution was set to Kolmogorov-Smirnov normality test with Dallal-Wilkinson-Lilliefor P value and create a QQ plot was selected. These data analyses pertain to data generated as described in 2.9.
Data analysis for selectivity included using Skyline to integrate peak areas and subsequently export normalized peak areas as an excel file. To calculate the slope of the line for the three-point response curve for each of the six biological matrices independently, peak areas were pasted into GraphPad Prism and the group analysis function was used to perform a linear regression analysis. The calculated equations of the curves were used to average across all six slope values which was then used to determine whether each of the individual slopes were within 10% of the average, in other words a (greater than) 10% difference was used to identify interference 31.
2.1. Patient and Blood Specimen Collection
The certificate of ethical approval for harmonized minimal risk clinical study pertaining to this project/study is active and available via the Board of Record REB Number: BC20-0440 through the University of Victoria Human Research Ethics Board Michael Williams Building, R. B202 PO Box 1700 STN CSC Victoria, BC V8W 2Y2 Tel: 250-472-4545. Blood specimens were collected from N = 10 individuals at the Victoria Lipid Clinic Society (Victoria, BC, Canada) by a registered nurse via venipuncture into lithium heparin coated blood collection tubes. A total of ten specimens were collected over a 91-day time period. Blood specimens were stored at 4˚C until processed within 72 h of blood collection.
2.2 Limiting-Dilution and RBC Spotting
Whole blood was used to aliquot 100 µl into 1.5 ml microcentrifuge tubes and centrifuged at 300x g for 5 min. The supernatant was pipetted out and discarded and packed RBCs were resuspended in 100 µl of 6.67% human serum albumin (HSA) (Sigma-Aldrich, Canada, SKU: A9731), which was previously prepared by dissolving 66.7 mg lyophilized powder in 1 ml LC-MS grade water and syringe filtered using a 0.22 µm filter. Limiting dilution (LD) was performed in a two-fold dilution series in 6.67% HSA using 384-well deep well small volumeTM microplate, polypropylene (Greiner Bio-One, Germany, REF: 784201). In detail, 10 µl of 6.67% HSA was first aliquoted per well, vertically from row A to K, the final dilution (in row K) being 2048-fold. Aiming to isolate 5-25 RBCs per spot, 0.25 µl from diluent wells (in the LD plate) were sampled using a 0.1-2.5 µl manual pipettor, and delivered into a low volume microscopy compatible 384 well plate (high content imaging plate, with lid black with clear 127 µm bottom 384 well, tissue culture treated cyclic olefin co-polymer, Corning, USA, REF: 4681), hereafter referred to as one-pot-384 (OP384). Sample spotting was performed by (reverse) capillary action, rather than dispensing with the pipettor plunger, by contact of the pipette tip with the bottom of the well for 10 sec. Care was taken not to apply a downward force on the pipettor as that could damage the 127 µm film thereby interfere with microscopy. Resting the weight of the pipettor while ensuring perpendicular contact with the surface was sufficient to achieve spotting/reverse capillary flow. The same pipette tip was used for spotting in the subsequent two wells at which point the pipette tip was discarded and the procedure was repeated until the number of desired replicates (N=10 per specimen) were spotted. In this way, the volume of sample per well was estimated at 80 nl (250 nl sample used to spot 3 wells). Wells were examined using an inverted phase contrast microscope at 100x total magnification (Bausch and Lomb PhotoZoom 31-19-14 Inverted Phase Contrast Microscope) to view erythrocytes encapsulated in (dried) albumin. The plate was stored at 4˚C until sample preparation.
2.3 CellenONE Assisted RBC Spotting
This was a pilot experiment to determine the capability of the PRM assay for measuring peptide targets in single RBCs and to assess response when the RBC input ranged from one to five cells. Spotting of RBCs directly into OP384 was performed at the Cellenion laboratory (Cellenion SASU, Lyon, France) using a cellenONE X1 instrument software version 2.0.0.984. For these experiments the concentration of HSA was lowered from 6.67% to 2.2% as the former reduced the stability of the droplet formation created by the proprietary dispensing technology; sciDROP PICO. The following parameters were used to isolate and dispense RBCs directly into OP384: Nozzle voltage 96 V, pulse duration 49 µsec, frequency 500 Hz and LED delay 200 µsec. For imaging, detection and isolation the background was set to ejection of 344 pix, sedimentation 180 pix, detection parameters were lower grey 10 upper grey 255, minimum diameter 6.5 µm, max diameter 100 µm and elongation 4. The isolation parameters were set to lower grey 10, upper grey 255, minimum diameter 7.5, max diameter 16.3 and elongation 1.65. Channel was transmission, selection mode was positive and threshold 10-255.
RBCs were deposited in OP384 plate as single RBCs, two RBCs, three RBCs, four RBCs, five RBCs and as HSA without RBC. The OP384 plate containing the deposits was then shipped to the University of Victoria Genome BC Proteomics Centre for subsequent sample processing (described in 2.4) and LC-MS analyses (described in 2.5).