Fig. 6: Micro-protocol for cell surface proteomics.

A Schematic of micro sample preparation method using an InStageTip approach for all steps after surface glycoprotein biotinylation on live cells. B Quantitative comparison of LFQ intensity for identified proteins using 30e6 cells in the standard, macro-protocol, versus 1e6 cells using our micro-protocol. LFQ values averaged from n = 2 biological replicates per preparation method; performed with RS4;11 B-ALL cells. C The micro method demonstrates excellent reproducibility across both biological replicates at 1e6 cell input. D Pearson R values of indicated cell inputs of RS411, AMO1, or primary myeloma against the 25e6–30e6 proteomic sample from the same cell line or primary sample. Data points for AMO1 and RS411 represent biological replicates compared to one of the 25e6–30e6 cellular input biological replicates. For primary myeloma, one primary sample MM1 was titrated at inputs of 25e6, 10e6, 5e6, 1e6, 0.5e6, and 0.1e6. E Total number of cell membrane-associated proteins identified using the micro-method at various cell inputs, on AMO1, RS411, and CD138+ tumor cells isolated from four relapsed/refractory myeloma patients. Samples underlying datapoints in E are from the same samples used for correlation analysis in D, with the addition of the 25e6–30e6 biological replicate for AMO1, RS411, and MM1 used for correlation as well as three additional primary myeloma patient samples (MM2, MM3, MM4) which were collected with total cellular inputs between 2e6–5e6. For B–E, source data are provided as a Source data file. F Quantitative comparison of identified cell membrane proteins in the 25e6 cell input primary sample (x-axis) versus averaged over the four profiled myeloma cell lines (y-axis). Pearson R reported. Source data available in Supplementary Data 1 and 7. G Cell membrane protein intensities in the MM1 sample with 25e6 input. Relevant therapeutic targets and other antigens noted in the manuscript are specifically labeled. H Immuno-targets and biomarker candidates identified using micro scale proteomics on the surface of CD138+ myeloma cells isolated from four patient samples. MaxQuant iBAQ absolute quantification intensity reported. For G, H, source data in Supplementary Data 7. I Schematic of primary myeloma vs. B-cell TMT proteomics experiment. Micro protocol was performed CD138+ cells isolated from an additional five primary myeloma patient samples and B-cells isolated from five healthy donors. Peptides were labeled with TMT-10plex reagents and combined prior to fractionation and LC-MS/MS. J PCA of myeloma and B-cell primary proteomic samples from TMT multiplex. K Comparison of Myeloma and B-cell membrane associated proteomes validates GGT1, ICAM3/CD50, ICAM1/CD54, and LY9 as some of most upregulated primary myeloma surface proteins relative to B-cells (log₂-fold change > |1 | ; p < 0.05 in blue). For J, K, source data in Supplementary Data 10. For boxplots in D, E, upper and lower hinges correspond to 25 and 75 percentiles, upper and lower whiskers extend to highest and lowest values within 1.5* IQR of the hinge, small-sized points indicate means, and center line corresponds to the median.