Urea (#U0631), Trizma base (#T2694), citric acid (#C0706), dimethyl sulfoxide (DMSO, #D2438), EDTA (#E7889), EGTA (#E0396), and iodoacetamide (IAM, #A3221) were obtained from Sigma (St. Louis, MO). Acetonitrile (MeCN, #A955), water (#W6, LCMS Optima^® grade), trifluoroacetic acid (TFA, LC-MS grade, #85183), tris(2-carboxyethyl)phosphine (TCEP, #77720), phosphate buffered saline (PBS, #BP-399-20), ammonium bicarbonate (A643-500), xylene (#422685000), and (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate) (CHAPS, #28300) detergent were obtained from Thermo Fisher Scientific (Waltham, MA). Rapigest (#186001861) was obtained from Waters (Milford, MA). Formic acid (#1.11670.1000) was obtained from EMD Millipore (Billerica, MA). Lys-C (Wako, #129-02541) and sequencing grade trypsin (#V5111, Promega, Madison, WI) were used for digestion of samples. Rabbit monoclonal antibodies were produced with Epitomics/Abcam (Cambridge, MA) and Excel Biopharm (Burlingame, CA). Mouse monoclonal antibodies were produced with Precision Antibody (Columbia, MD) and the Antibody Development Facility at the Fred Hutchinson Cancer Research Center (Seattle, WA). Light (unlabeled) synthetic peptides were obtained from Vivitide (Gardner, MA) as crude (flash purified) grade. Cleavable stable isotope-labeled (heavy) peptides from Vivitide corresponding to the tryptic analyte sequence were purified >95% by HPLC, labeled with [¹³C and ¹⁵N] at the tryptic C-terminal Arg or Lys, and quantified by amino acid analysis (AAA). Aliquots of peptide standards were stored in 5% acetonitrile/0.1% formic acid at −80°C until use.

HeLa (American Type Culture Collection (ATCC), #CCL-2), Jurkat (ATCC, #TIB-152), A549 (ATCC, #CCL-185), MCF7 (ATCC, #HTB-22), and NCI-H226 (ATCC, # CRL-5826) cell lines were cultured and harvested according to manufacturers’ specifications. Briefly, cells were sub-cultured and after trypsinization, were centrifuged at 1500 rpm for 6 minutes and supernatant was removed and discarded. Cells were washed once with 1X PBS, centrifuged as before, resuspended in 1X PBS, and counted. Cells were subdivided according to desired cell number in 15 mL centrifuge tubes and centrifuged as before. Cell pellets were frozen and stored at -80°C until whole cell lysis. Cell pellets were lysed using RIPA lysis and extraction buffer (Thermo Fisher Scientific, # 89900) following the manufacturer’s protocol. Mammalian Protease Inhibitor (VWR, #VWRVM250) was added according to manufacturer’s instructions. Lysate protein concentration was estimated with a BCA assay (Pierce, #23225) as described by the manufacturer’s instructions.

FFPE tissue and plasma samples used for fit-for-purpose validation studies were commercially acquired from BioIVT (Westbury, NY). Frozen tissue and plasma samples for determination of detectability were supplied by the Clinical Proteomics Tumor Analysis Consortium (CPTAC) as anonymized samples from consenting donors collected under IRB-approved protocols. FFPE tissue was supplied by the Veteran’s Administration from consenting donors collected under IRB-approved protocols. Tissue sub-compartment cellularity (e.g. tumor, stroma, adipocytes, lymphocytes) was calculated using the HALO Tissue Classifier (Indica Labs, NM) using a machine learning algorithm that was trained to apportion the total area of a section into the following components: epithelium, stroma, adipocytes, lymphocytes, and red blood cells; the training was based on results from two pathologists using blinded stained slides from breast cancer specimens (40).

To produce lysates for immuno-MRM analysis, frozen tissue was cryopulverized in a cryoPREP CP-02 (Covaris, Woburn, MA) and stored frozen until analysis. 5 μL of lysis buffer (25 mM Tris, 6 M Urea, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Sigma protease inhibitor (#P8340), 1% (v/v) Sigma phosphatase inhibitor cocktail 2 (#P5726), 1% (v/v) Sigma phosphatase inhibitor cocktail 3 (#P0044)) was added for each mg wet tissue weight (up to 1000 μL). The sample was vortexed for 10-15 sec and sonicated three times in a cup horn probe (filled with ice water) at 50% power for 30 seconds. The samples were stored in liquid nitrogen until the day of digestion.

FFPE samples were processed as described previously (48). Briefly, slide-mounted FFPE tissue sections were placed in a 4 slide holder. The slides were incubated three times in xylene for 3 min followed by 100% (v/v) ethanol twice for 3 min. The tissue was then hydrated twice in 85% (v/v) ethanol for 3 min, 70% (v/v) ethanol for 3 min, and distilled water for 3 min. The tissue was then blotted and scraped off the slide into a screw cap microfuge tube. To each sample tube (containing three FFPE 10 µm tissue sections), extraction buffer (0.2% RapiGest in 50 mM ammonium bicarbonate, NH₄HCO₃) was added and incubated at 95°C for 30 minutes with mixing at 1000 rpm (Thermomixer, Eppendorf, Enfield, CT). The samples were then cooled on ice for 5 minutes and sonicated twice in a cup horn probe (filled with ice water) at 50% power for 30 sec. The samples were then incubated at 80°C for 120 minutes with mixing at 1000 rpm and then cooled on ice for 5 min. 100 μL of 50 mM NH₄HCO₃, pH 8.0 was added, and the samples were sonicated twice in the cup horn probe (filled with ice water) at 50% power for 30 sec. Following processing, all samples were stored at -80°C until the day of digestion.

Protein concentrations of lysates were measured in triplicate using Micro BCA Protein Assay Kit (Pierce, #23235).

A mix of cleavable stable isotope-labeled peptide standards was added to the lysate at 200 fmol/capture. 500 μg frozen tissue lysates or 15-270 μg of protein (FFPE) was transferred to a deep-well plate for processing on an EpMotion 5057 (Eppendorf). Lysates were reduced in 30 mM TCEP for 30 minutes at 37°C with shaking, followed by alkylation with 50 mM IAM at room temperature. Lysates were then diluted with 0.8 mL 200 mM TRIS before Lys-C endopeptidase was added at a 1:50 enzyme:substrate ratio by mass and incubated for 2 hours at 37°C with mixing at 600 rpm (Thermomixer, EpMotion 5057). After 2 hours, trypsin was added at a 1:50 enzyme:substrate ratio. Digestion was carried out overnight at 37°C with mixing at 600 rpm. After 16 hours, the reaction was quenched with formic acid (final concentration 1% by volume).

Plasma (2 x 50 μL aliquots of each sample) was denatured with 150 μL of lysis buffer (25 mM Tris, 6 M Urea, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Sigma protease inhibitor (#P8340), 1% (v/v) Sigma phosphatase inhibitor cocktail 2 (#P5726), 1% (v/v) Sigma phosphatase inhibitor cocktail 3 (#P0044)). The plasma was reduced with 30 mM TCEP at 37°C for 30 min, and alkylated with 50 mM IAM at room temperature for 30 min. Urea concentration was diluted 10-fold with 200 mM Tris prior to overnight digestion at 37°C with Lys-C/trypsin using a 1:50 (w/w) enzyme:substrate. Digestions were terminated with formic acid.

The mixture was desalted using Oasis HLB 96-well plates (Waters #WAT058951) and a positive pressure manifold (Waters #186005521) according to the following procedure: wash cartridge with 4 × 400 μL of 50% acetonitrile in 0.1% formic acid, equilibrate with 4 × 400 μL of 0.1% formic acid, load total volume of digest, wash with 4 × 400 μL of 0.1% formic acid, and elute with 3 × 400 μL of 50% acetonitrile in 0.1% formic acid. The eluates were lyophilized and stored at -80°C.

Enrichment was performed as previously described (49) with the following modifications. The final assay consisted of a mixture of 50 antibodies. Antibodies were crosslinked on protein G beads (GE Sepharose, #28-9513-79), and peptide enrichment was performed using 1 μg antibody - protein G magnetic beads for each target. Trypsin-digested lysate was resuspended in 200 μL 1X PBS + 0.01% CHAPS (pH was adjusted to 7.0 with 10 μL of 1 M Tris, pH 9). For plasma, the two digestion aliquots were combined after resuspension to a total volume of 200 μL. Beads were mixed in the incubation plate, washed twice in 1X PBS buffer + 0.01% CHAPS, washed once in 1/10X PBS + 0.01% CHAPS, and peptides were eluted in 26 μL of 5% acetic acid/3% acetonitrile/50 mM citrate. The elution plate was covered with adhesive foil and frozen at -80°C until analysis.

LC-MS was performed with an Eksigent 425 nanoLC system with a nano autosampler and chipFLEX system (Eksigent Technologies, Dublin, CA) coupled to a 5500 QTRAP mass spectrometer (SCIEX, Foster City, CA). Peptides were loaded on a trap chip column (Reprosil C18-AQ, 0.5 mm x 200 μm, SCIEX, #804-00016) at 5 μL/min for 3 minutes using mobile phase A (0.1% formic acid in water). The LC gradient was delivered at 300 nL/minute and consisted of a linear gradient of mobile phase B (90% acetonitrile and 0.1% formic acid in water) developed from 2-14% B in 1 minute, 14-34% B in 20 minutes, 34-90% B in 2 minutes, and re-equilibration at 2% B on a 15 cm x 75 μm chip column (ChromXP 3C18-CL particles, 3 μm, SCIEX, #804-00001). The nano electrospray interface was operated in the positive ion MRM mode. Parameters for declustering potential (DP) and collision energy (CE) were taken from optimized values in Skyline. Scheduled MRM transitions used a retention time window of 210 seconds and a desired cycle time of 1.5 seconds, enabling sufficient points across a peak for quantification. A minimum of two transitions per peptide, including endogenous and spiked heavy peptides, were recorded for each light and heavy peptide.

MRM data acquired on the 5500 QTRAP were analyzed by Skyline (50, 51). Peak integrations were reviewed manually, and transitions from analyte peptides were confirmed by the same retention times and relative transition areas of the light peptides and heavy stable isotope-labeled peptides. Transitions with detected interferences were not used in the data analysis. Integrated raw peak areas were exported from Skyline and total intensity was calculated using Peak Area + Background. Transitions were summed for each light/heavy pair and peak area ratios were obtained by dividing peak areas of light peptides by that of the corresponding heavy peptides (or vice versa for response curves). All measurements were filtered by the LLOQ (i.e., all measurements were required to be above the LLOQ). Peak area ratios were log (base 2) transformed for statistical analysis.

Quantitative RNA sequencing data (FPKM) was available for 74/110 frozen tissues (52). MRM peak area ratios were log (2) transformed and correlated with gene expression data using Pearson regression. Correlations were done within each tumor type and across all tumor types (n = 74).

Minimum tissue requirements were calculated by using frozen tissue results of signal-to-noise levels measured at 500 μg input. The signal-to-noise levels were compared to the LOQ for each analyte using a one-sample t-test and those analytes with 95% confidence above LOQ were further converted to input mass using linear scale dilution. The confidence interval in the minimum tissue calculation was determined based on the standard deviation of signal-to-noise distribution and degrees of freedom (equal to the number of samples for each tissue site -1).

Four experiments (described below) were performed to characterize the analytical performance of the assays: i) digestion time course for stable recovery of peptides, ii) response curves, iii) repeatability, and iv) stability.

Recombinant proteins and over-expressed lysates were obtained from Origene (Rockville, MD) or Novus Biologicals (Littleton, CO), see Supplementary Table 1 for catalog numbers. Western immunoassays were performed using traditional immunoblotting techniques according to the designed protocol described in the CPTAC Antibody Portal (antibodies.cancer.gov) SOP M-103 with the following modifications. Proper sample loading on the 4-20% Criterion TGX Stain-free precast gels (Bio-Rad, # 5678094) was verified by rapid florescence detection with ChemiDoc MP imager. Traditional immunoblotting used 10 μg/mL recombinant protein under reducing conditions (20 mL, 200 ng total protein/lane). Whole cell lysates were diluted to 2.5 mg/mL in reducing conditions (20 mL, 50 μg total protein/lane). Transfers of protein from Bio-Rad precast gels were performed by Bio-Rad Turbo-Blot at “High MW” setting for 10 minutes. Blocking of the membrane was performed using Bio-Rad Blotting Grade Blocker (Bio-Rad, #1706404) at 5% in 1X PBS/0.5% Tween-20. Primary antibodies (1 mg/mL) were diluted in 1X PBS/0.5% Tween-20 to a dilution of 1:5000 at a total volume of 25 mL. Washing of membrane was conducted using 1X PBS/0.5% Tween-20 three times. Secondary HRP-linked rabbit specific antibody (Jackson ImmunoResearch, West Grove, PA, 111-035-144) or secondary HRP-linked mouse antibody (Jackson Immunoresearch Laboratories 115-035-062) was diluted at 1:5000 in 1 X PBS/0.5% Tween-20 at a final volume of 25 mL. Immuno-detection was performed using colorimetric substrate Opti-4-CN (Bio-Rad, #1708235) at 1 mL per blot or using enhanced chemiluminescence using Clarity Western ECL Substrate (BioRad, #1705061) at 1 mL per blot. Development of immunoblot was captured using Bio-Rad ChemiDoc MP imaging system.

The Simple Western (Wes, ProteinSimple, San Jose, CA, #004-600) system was used to detect primary antibody binding to a target protein in cell lysates (MCF10A, LCL57). The Simple Westerns were performed following the procedures detailed in the CPTAC Antibody Portal (antibodies.cancer.gov) SOP M-134 with the following modifications. Cell lysates were run using the 12-230 kDa separation module, 8 x 25 capillary cartridges (ProteinSimple, #SM-W004), and detected with Anti-Rabbit Detection Module (ProteinSimple, #DM-001) or Anti-mouse Detection Module (ProteinSimple, #DM-002). Cell lysates were run at a concentration of 200 μg/mL and incubated with primary antibodies diluted 1:500.

Protein array analyses were performed using NCI-60 cell lines obtained from the Cancer Research Technology Program at NCI-Frederick (Frederick, MD). The NCI-60 cell lines were collected at the log phase growth and protein prepared by resuspending cell pellets in RIPA (Thermo Fisher Scientific, #89900) per the manufacturer’s recommendations; total protein concentration was measured by BCA Protein Assay kit (Thermo Fisher Scientific, #23225). Quantification of protein expression values was performed by well-based reverse phase protein array (RPPA) as previously reported (53, 54). Briefly, five microliters (100 ng/well) of NCI-60 cell line antigens in PBST (1X PBS, 0.1% Tween-20) were applied onto 96-well Multi-Array™ plates (96 HB SECTOR Plate, Meso Scale Discovery, Gaithersburg, MD). The plates were allowed to dry at room temperature for 2 hours. Prior to primary antibody incubation, the antigen-coated plates were blocked with 5% non-fat dry milk in PBST for 1 hour at room temperature. Target-specific antibodies were diluted (1:1000 to 1:5000) with 5% BSA in PBST. For each cell line, 25 μL of antibody were added and incubated overnight at 4 °C. The plates were washed with PBST and followed by a 90 min incubation with goat anti-mouse or anti-rabbit SULFO-TAG™ antibodies (Meso Scale Discovery) at a dilution of 1:2000 (0.5 µg/mL) containing 5% non-fat dry milk in PBST. Plates were washed, and MSD-T read buffer was added to the plate to detect binding signals using MESO QuickPlex SQ 120 reader (Meso Scale Discovery). PBST-coated wells were included on each plate as a control of non-specific binding. For each mAb, the electrochemical luminescence value of each cell line was normalized by the average value of the 60 cell lines. After normalization, levels below 0.5 were interpreted as weak or negative expression and levels above 1.5 were interpreted as strong, positive expression.

Targeted mass spectrometry data are available in the supplementary tables and Panorama Public (55), a public repository of targeted proteomics experiments (https://panoramaweb.org/IO1immunoMRM.url. Characterization data for assays can be found via the CPTAC Assay Portal (https://assays.cancer.gov) and antibodies are available through the CPTAC Antibody Portal (antibodies.cancer.gov) (see Supplementary Table 2 for IDs).

Because there are no adequate predictive or prognostic biomarkers to guide delivery of immunotherapies, there is great interest in high quality assays to quantify immunomodulatory proteins for correlative studies in clinical trials in hopes of identifying single or panels of biomarkers showing clinical utility (56). Proteins and phosphorylation sites related to immunomodulatory functions were identified as targets for assay development through literature searching and expert consultation. A panel of experts in immuno-oncology was convened from academia and industry to nominate immunomodulatory proteins of interest for assay development ( Table 1 ) to support correlative studies to identify novel biomarkers or response or immune-related adverse events. Stoichiometric surrogate peptides for the target proteins were identified using established procedures (88, 89), by mining liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic and phosphoproteomic datasets (16, 17, 90–98) and public databases (52, 99, 100) for empirical evidence of LC-MS/MS detectability. Peptides were ranked based on the intensity and frequency of empirical observations in the datasets, chemical properties (e.g., amino acid composition, hydrophobicity), and physical properties (e.g., length, position in protein). Finally, peptides with frequent mutation sites [contained in dbSNP (101)] and PTMs [PhosphoSitePlus (102)] were avoided.

A list of 55 peptides (including multiple peptides per protein) were selected for assay development, representing 46 proteins and including 5 phosphorylation sites. Peptide sequences selected for assay development are listed in Table 1 . Unlabeled (i.e., light) tryptic peptides were synthesized for assay development, and cleavable heavy stable isotope-labeled peptides were synthesized for use as internal standards. The cleavable standards incorporate 2-5 additional amino acids (corresponding to the native protein sequence) on either side of the tryptic cut sites. This strategy was adopted in order to spike the standards into the sample prior to digestion and provide a within-sample control for trypsin digestion (20). The synthetic light peptides were used to optimize the selection of specific precursor and fragment ion pairs (i.e., transitions), determining the chromatographic retention time of each peptide, and optimizing collision energy parameters in the mass spectrometer ( Supplementary Table 3 ).

Immuno-MRM uses anti-peptide antibodies (103) for enrichment prior to LC-MRM and is applicable for quantifying expression of high and low abundance proteins as well as phosphopeptides ( Figure 1A ). For the immuno-MRM assay panel, we generated custom anti-peptide monoclonal antibodies to the linear peptide sequences using established approaches (25, 26). We used 50 custom monoclonal anti-peptide antibodies ( Supplementary Table 2 ) for the multiplexed immuno-MRM assay, including 35 antibodies generated for this study and 15 antibodies from previously characterized assays (26, 40).

Trypsin digestion has been identified as a source of variability in sample processing for targeted proteomics (29, 104). Therefore, to achieve reproducible digestion, we sought to identify a timepoint where release of the tryptic peptides (light and heavy) was most stable and at a maximum. Recovery of peptides following trypsin digestion was characterized using a time course digestion study using an optimized procedure incorporating Lys-C and trypsin for digestion (105). Aliquots of pooled cell line lysates were spiked with heavy peptides and digested according to the methods described herein. To evaluate the release of endogenous tryptic peptides over time, aliquots were taken after 1, 6, 16, and 24 hours of incubation with trypsin and subjected to immunoaffinity enrichment and analysis by LC-MRM. The recovery of light peptides reached a stable maximum at the 16-hour timepoint, with two peptides showing slightly lower (~20%) signals ( Figure 1B ). To achieve the maximum recovery for most peptides in the assay, we used overnight (i.e., 16 hours) digestion for processing samples.

Performance of the multiplexed IO-1 immuno-MRM assay panel was characterized in tissue and plasma matrices according to published guidelines (47, 106) to establish figures of merit including linearity, limits of quantification, repeatability, and stability. The linear ranges and limits of quantification (LOQs) were determined by response curves using pooled background matrices of protein lysates from i) frozen lung tumor tissues, ii) formalin fixed and paraffin embedded (FFPE) lung tumor tissues, and iii) plasma. Aliquots (150 µg) of the pooled tissue lysates or 10 µL aliquots of plasma were spiked with synthetic cleavable heavy stable isotope-labeled standards and serially diluted to yield concentrations of 2000, 200, 20, 8, 3.2, 1.28, 0.512, 0.205, and 0 (blank) fmol/mg or fmol/µL, respectively, and each concentration level was digested in process triplicate. Synthetic light tryptic peptides were added at a constant concentration of 200 fmol/mg or 200 fmol/µL prior to desalting the digested peptides. The monoclonal antibodies were coupled to magnetic beads and used to enrich the peptides. The eluates were analyzed by LC-MRM-MS. Peptide specificity was confirmed by equivalent retention times and relative transition areas of the heavy and light peptides.

Peak area ratios (heavy:light) were plotted as a function of analyte concentration to determine the assay figures of merit in the response curves. Figures 1C, D show representative response curves measured in tissue and plasma. Lower limits of quantification (LLOQs) were determined by the lowest point with CV < 20%. Linear ranges were determined by using points on the linear regression with correlation coefficients greater than 0.9. For assays where the highest concentration point was still linear, the linear range was a minimum estimate. Figures of merit are reported for each peptide in Supplementary Table 4 . Median linear dynamic range was ≥3.2 orders of magnitude in all matrices with median LLOQ 12.8 fmol/mg (range 2-2000 fmol/mg) in tissues and 0.128 fmol/µL (range 0.02-2000 fmol/µL) in plasma. The characterized LLOQs and linear ranges were the same for frozen and FFPE tissues ( Supplementary Figure 1 ). Three peptides (LIME1.SSTC[cam]GAGT[ph]PPASSC[cam]PSLGR, LIME1.SSTC[cam]GAGTPPASSC[cam]PSLGR, and VSIR.GHDVTFYK) exhibited low signal in the curves, likely due to poor antibody activity when cross-linked to the beads, and failed to produce adequate linearity for further use.

Intra-assay (within day) and inter-assay (between day) repeatability were determined by performing complete process triplicate measurements for the multiplexed assay at three concentrations of spiked peptides over 5 days. Heavy cleavable peptide standards were spiked into 150 µg aliquots of the pooled FFPE tissue lysate or 10 µL aliquots of plasma matrix at three concentrations (10, 100, 1000 fmol/mg; low, medium, high) with addition of equal amounts of light peptides (200 fmol/mg) to each aliquot. Specificity was confirmed using the same criteria as described above. For the assays measured in tissue matrix, the median intra-assay variability was 7.5%, 5.4%, 3.5% for low to high concentration samples and the median inter-assay variability was 9.5%, 10.8% and 11.8% (low to high) ( Figure 1C and Supplementary Table 5 ). Two outlier assays (ATM.NLS[ph]DIDQSFNK and ATM.NLSDIDQSFNK) showed high intra- or inter-assay variability in tissue samples due to signal intensities below the LOQ at the lowest concentration values. For the assays measured in plasma matrix, the median intra-assay variability was 13.6%, 5.8%, 4.5% from low to high concentration samples and the median inter-assay variability was 24.9%, 14.9% and 16.5% from low to high concentration samples ( Figure 1D and Supplementary Table 5 ). Three assays (ANXA1.AAYLQETGKPLDETLK, PTPRC.LFLAEFQSIPR, PSMA1.ETLPAEQDLTTK) showed high variability when applied to plasma. High variability was likely due to different factors for the peptides. ANXA1.AAYL had high variability in the heavy signal, PTPRC.LFLA was likely due to instability of the peptide following processing (see below), and PSMA1.ETLP had one day with outlier values compared to other days.

To characterize expected variability in measuring endogenous proteins, heavy cleavable peptide standards were spiked into 150 µg aliquots of a pooled FFPE lung tissue lysate and 10 µL aliquots of plasma. Measurements were made using 5 complete process replicates of endogenous peptide over 5 days (n=25) ( Figures 1C, D ). Forty-one peptides were detected above LLOQ in the endogenous tissue sample. The median intra-assay variability for endogenous detection was 5.2% (range 2.4-16.9%) and the median inter-assay variability was 6.9% (range 0.8-21.4%). Twenty peptides were detected above LOQ in the endogenous plasma sample. The median intra-assay variability was 12.2% (range 4.1-34%) and the median inter-assay variability was 21% (range 4.5-62%).

Peptide stability was evaluated by spiking heavy peptide (200 fmol/mg) into 150 µg aliquots of the pooled FFPE tissue lysate or 200 fmol/µL into 10 µL aliquots of plasma matrix, followed by digestion and immunoaffinity capture. The samples were analyzed after storage under four conditions: (i) 4°C on the autosampler for 8 h, (ii) 4°C on the autosampler for 48 h, (iii) after two freeze-thaw cycles, and (iv) -80°C for 5 weeks; control samples were analyzed immediately. Each test case was measured in triplicate. The variability (%CV) and percent difference, comparing peak area ratio between control samples and samples with different handling conditions, were used to evaluate peptide stability. The median %CV for the test samples ranged between 2.5-8.5% in FFPE and 3.1-6.5% in plasma ( Supplementary Table 6 ), within expectations based on repeatability measurements ( Supplementary Table 5 ) for both matrices. The median percent difference relative to the fresh sample ranged 0.1-3.5% in FFPE and 2.0-5.6% in plasma, indicating acceptable stability for the peptides ( Figures 1C, D and Supplementary Table 6 ). In FFPE, three assays (CCL5.EYFYTSGK, ATM.NLS[ph]DIDQSFNK, CD274.NIIQFVHGEEDLK) showed high variability and/or relative difference, indicating these peptides should not be allowed to sit on the autosampler for longer than 8 hours. Likewise, three assays (LGALS1.SFVLNLGK, CD274.NIIQFVHGEEDLK, PTPRC.LFLAEFQSIPR) showed high variability or percent difference in plasma samples after storage on the autosampler, indicating these peptides should be analyzed by LC-MRM within 8 hours.

We next applied the multiplex assay to a panel of tumor tissue specimens with two aims: (i) evaluate the utility of the assay for measuring endogenous levels of analytes in clinical biospecimens, and (ii) determine sample requirements for analyte detection in tissue where clinical material may be limited. The tissue panel included 110 frozen and 25 FFPE biospecimens collected from 12 different tumor types.

Frozen tissue biospecimens were obtained from 11 tumor types including brain, breast, colorectal, endometrium, head and neck, kidney, lung (squamous cell carcinoma and adenocarcinoma), ovarian, pancreas, and soft tissue sarcoma ( Figure 2A ). The distribution of cellular heterogeneity was evaluated using the H&E-stained slides, and a supervised algorithm (40) (HALO) was used to assign the fraction of tissue area pertaining to predominant cell types (e.g., adipose, lymphocytes, red blood cells, stroma, and tumor). The distribution of these cellular subtypes is plotted in Figure 2B for the frozen tumors. Overall, most of the biospecimen area can be assigned to tumor and stromal cells, with small proportions of adipose, lymphocytes, and red blood cells. Notably, ovarian tumors contained the highest fraction of adipocytes. These fractions show that the tumors were relatively good quality specimens, with median percentage of tumor cells >50%.

To determine the ability of the IO-1 immuno-MRM assay to measure endogenous proteins, we used 500 µg aliquots of the frozen tissue lysates as input. Each biospecimen was independently digested, enriched for the IO-1 analytes, and analyzed by LC-MRM. Specificity of endogenous peptide detection was assured by equal retention times and relative areas of light and heavy transitions. Overall, 48/52 peptides corresponding to 45 proteins were detected above LLOQ in the frozen tissue biospecimens. Four peptides were not detected in the biospecimen panel: phospho-ATM pS367, phospho-ATM pS2996, phospho-FAS pY291, and unmodified TNFRSF9. Most peptides were detected in all samples, with a small number of peptides detected below the LLOQ in a subset of samples (see Figure 2C ). The median inter-individual CV was 77% (range 32-676%). This variation is likely due to differences between the individual tumors and not differences in the tissue type, since unsupervised clustering of the expression levels for the peptides in the IO-1 multiplex panel ( Figure 2D ) did not separate the samples by location. This is not surprising, since these proteins were not chosen for site-specific differences in expression, but rather for their role in immunomodulatory functions, which may vary from tumor to tumor. Finally, correlation of expression levels with mRNA (17, 52, 90–97, 107) showed a median correlation coefficient of 0.56 ( Figure 2E ), consistent with previous reports measuring protein-RNA correlation (15–17).

Given that clinical biospecimens often have limited available material, we next sought to determine the sample requirements for detection of endogenous protein in tissue to use as a guide for expectations in future studies. We used the results of the frozen tissue array ( Figure 2 ) to estimate the minimum amount of tissue needed for the analyte to remain above the LOQ using the distribution of signal-to-noise values measured in tumor samples, compared to the LLOQ characterized in tissue matrix. Figure 3 shows the number of peptides predicted to be detected for tissue inputs ranging from 10-500 µg. Notably, the number of peptides detected as the amount of tissue decreases remains within ~80% of total, even with ten-fold less input. This indicates that the assays are amenable to a range of biospecimen sizes and input amounts.

To evaluate the potential for applying the assays to retrospectively collected biospecimens, we evaluated the detection of peptides in FFPE tissues representing 5 tumor types, including colorectal, head and neck, lung (squamous cell carcinoma and adenocarcinoma), and prostate ( Supplementary Figure 2 ). Optimized sample processing conditions were used to process the samples (48), however the limited material available for these samples resulted in sample inputs ranging from 2 to over 10-fold less material compared to frozen tissue biopsies. Despite these limitations, the assay successfully quantified 33/52 peptides above LOQ in more than half of samples ( Supplementary Figure 2 ).

To evaluate the ability of the assay to measure protein levels in plasma, we applied the assays to a panel of 45 plasma samples from patients with 3 different tumor types (breast, colon, ovarian) ( Figure 4A ). We used 100 µL aliquots of plasma as input for the assay. Each specimen was independently digested, enriched for the IO-1 analytes, and analyzed by LC-MRM. Specificity of endogenous peptide detection was assured by equal retention times and relative areas of light and heavy transitions. Overall, 38/52 peptides corresponding to 37 proteins were detected above LOQ in the plasma samples. The histogram of number of peptides detected across the panel showed that over half (n=24) of peptides were detected in all samples analyzed, with a small number of peptides detected below the LOQ in a subset of samples ( Figure 4B ) and 14 peptides not detected in any sample, likely due to the analytes not being secreted or shed to quantifiable levels. Compared to the variation seen in the tissue panel, there were less inter-individual differences in plasma, with the median inter-individual CV being 47% (range 10-152%). Like the tissue samples, unsupervised clustering of the expression levels for the peptides in the IO-1 multiplex panel ( Figure 4C ) did not separate the samples by tumor type.

Finally, there were 25 plasma samples for which we also analyzed tissue specimens from the same patient. The distribution of correlation coefficients for expression levels found in tissue and plasma from the same patient are plotted in Figure 4D . Overall, 31 peptides were detected in both tissue and plasma specimens, but there was poor intra-patient correlation between the two sample types. Based on previous results (108), we expected there may be high overlap in the peptides detected; however, it was unknown how the relative quantities may be correlated. Because protein abundance may also vary depending on specimen collection conditions (e.g., blood contamination in tissue specimens and/or cell shearing during plasma collection), we also examined if blood contamination in tissue specimens affected the correlation of peptides detected in tissue and plasma. A “blood contamination” score was assigned to each tissue specimen using the intensity of gel electrophoresis bands corresponding to the molecular weights of albumin and hemoglobin in each tissue lysate; however, correlation between tissue and plasma levels did not improve using only the samples with high “blood contamination” scores (data not shown).

In parallel to their development for immuno-MRM, the new antibodies generated for this study were characterized for reactivity to the target proteins in traditional immunoassay approaches. Antibodies were tested for utility in traditional Western blotting or Wes system using purified recombinant proteins (where available) and/or lysates from cancer cell lines. Blots were deemed positive if a prominent band at the expected molecular weight (given by the vendor for the recombinant proteins or based on the molecular weight reported in UniProt.org) was observed. For this set of antibodies, 47% (14/30) of the antibodies tested (14/30) were positive against recombinant proteins, and 60% (12/20) were positive against lysates (not all antibodies that were negative for the recombinant protein were tested against cell line lysates). These results were consistent with expectations based on previous work (109). Antibodies scoring positive in Western blotting were tested for application in protein array using the NCI-60 cell line array. Overall, 3 of the antibodies tested were positive, based on normalized levels above background. The data are summarized in Supplementary Table 7 . The monoclonal antibodies and characterization data are available as a resource for the research community via the CPTAC antibody portal (antibodies.cancer.gov; see Supplementary Table 2 ).