Sample Preparation Methods for Targeted Single-Cell Proteomics

Abstract

We compared three cell isolation and two proteomic sample preparation methods for single-cell and near-single-cell analysis. Whole blood was used to quantify hemoglobin (Hb) and glycated-Hb (gly-Hb) in erythrocytes using targeted mass spectrometry and stable isotope-labeled standard peptides. Each method differed in cell isolation and sample preparation as follows: 1) FACS and automated preparation in one-pot for trace samples (autoPOTS); 2) limited dilution via microscopy and a novel rapid one-pot sample preparation method that circumvented the need for the solid-phase extraction, low-volume liquid handling instrumentation and humidified incubation chamber; and 3) CellenONE-based cell isolation and the same one-pot sample preparation method used for limited dilution. Only the CellenONE device routinely isolated single-cells from which Hb was measured to be 540-660 amol per red blood cell (RBC), which was comparable to the calculated SI reference range for mean corpuscular hemoglobin (390-540 amol/RBC). FACSAria sorter and limited dilution could routinely isolate single-digit cell numbers, to reliably quantify CMV-Hb heterogeneity. Finally, we observed that repeated measures, using 5-25 RBCs obtained from N = 10 blood donors, could be used as an alternative and more efficient strategy than single RBC analysis to measure protein heterogeneity, which revealed multimodal distribution, unique for each individual.

Keywords: HbA1c; Single-cell proteomics; carboxymethyl hemoglobin; glycated hemoglobin; hemoglobin; one-pot; quantitative; red blood cell; targeted.

Figure 1

Consistency of peptide chromatographic peak areas, retention times, and identifications from nano-LC-MS analysis when samples were prepared with the detergent-free one-pot rapid digestion protocol. RBCs were spotted and counted in OP384 and processed using a detergent-free one-pot rapid digestion described herein. The input number of RBCs ranged from 0 to 8 RBCs with 0 RBCs serving as a background control. The X-axes denote the number of RBCs input. Panels A, B, and C depict peak areas and total ion chromatogram (TIC) normalized peak areas for trypsin and Lys-C derived HSA, HBB, and HBA peptides, respectively, and across all replicates. Panel D depicts peptide retention times for HSA (left), HBB (middle), and HBA (right) across all replicates; human serum albumin (HSA), hemoglobin beta subunit (HBB), hemoglobin alpha subunit (HBA).

Figure 2

Endogenous measure of peptide targets using near-single-cell and single-cell preparations. Panel A displays data generated at Site 2 using PRM to measure endogenous peptides prepared via the previously described autoPOTS workflow. RBCs were deposited directly into autoPOTS, in four replicates, aiming to deliver 0 (control background), 5, 50, and 250 RBCS which were subsequently processed for PRM using the autoPOTS workflow. The PRM chromatograms are representative of one of four replicate preparations and are labeled accordingly with the peptide targets. Data presented in panels B and C were generated at site 1 using PRM to measure endogenous peptide targets prepared with the one-pot rapid digestion protocol. Limiting dilution was used to isolate single RBCs in triplicate (RBC = 1 R1 through to RBC = 1 R3) along with triplicate control no RBC inputs (RBC = 0 R1 through to RBC = 0 R3). Single RBC isolation was confirmed via phase contrast microscopy at 100× magnification and can be identified in the images via the yellow arrow. Samples were prepared via one-pot rapid digestion and the resulting PRM chromatograms for both peptide targets are provided below the associated well (image) and labeled accordingly. Normalized peak area ratios (to CM-SIL and SIL spiked in at 2 fmol total) are displayed in panel C revealing significantly higher signals for peptide targets in wells containing single RBC compared to the no cell input wells.

Figure 3

Endogenous analyte concentration in RBCs isolated via CellenONE and one-pot rapid digestion sample preparation. RBCs were isolated directly into OP384 wells and the resulting droplets viewed under 100× magnification using phase contrast microscopy and images are displayed in panel A. Panels B and C display PRM chromatograms for CM-VHLTPEEK and VHLTPEEK peptides with total endogenous signal and CM-SIL and SIL peptide chromatograms overlaid. The CM-SIL and SIL peptides were spiked in at 2 fmol total. Panel D displays the scatter plot of the endogenous peptide signal as peak area ratio normalized to counterpart CM-SIL and SIL peptides plotted against the number of input RBCs.

Figure 4

One-pot rapid digestion isotope dilution PRM to measure RBC heterogeneity. The heights of the violin plots depict the variance across repeated measures while the shapes are indicative of (multi)modality. The solid line in each plot represents medians and dashed lines represent quartiles. The corresponding HbA_1c values, in order from individual 1 to 10, were: 6.0%, 6.5%, 5.4%, 6.3%, 6.4%, 8.1%, 6.0%, 6.6%, 7.8%, and 5.5%. The right panel depicts a scatter plot comparing %CM-VHLTPEEK measured via MRM in bulk sample preparations to the mean %CM-VHLTPEEK when measured in low input (5–25 RBCs) samples via PRM. Linear regression showed no correlation with an R-Squared value of 0.34.