Ultrasensitive single-cell proteomics workflow identifies >1000 protein groups per mammalian cell

Abstract

We report on the combination of nanodroplet sample preparation, ultra-low-flow nanoLC, high-field asymmetric ion mobility spectrometry (FAIMS), and the latest-generation Orbitrap Eclipse Tribrid mass spectrometer for greatly improved single-cell proteome profiling. FAIMS effectively filtered out singly charged ions for more effective MS analysis of multiply charged peptides, resulting in an average of 1056 protein groups identified from single HeLa cells without MS1-level feature matching. This is 2.3 times more identifications than without FAIMS and a far greater level of proteome coverage for single mammalian cells than has been previously reported for a label-free study. Differential analysis of single microdissected motor neurons and interneurons from human spinal tissue indicated a similar level of proteome coverage, and the two subpopulations of cells were readily differentiated based on single-cell label-free quantification.

Fig. 1. Single-cell proteomics workflow. Proteins from a single cell are extracted and digested, the resulting tryptic peptides are separated using a narrow-bore nanoLC column and ionized at an etched electrospray emitter. Singly charged ions are filtered using the FAIMS Pro interface and transmitted ions are detected using the Orbitrap Eclipse Tribrid MS.

Fig. 2. Proteome coverage for HeLa digest and single HeLa cells. (A) FAIMS method optimization using 0.5 ng aliquots of HeLa protein digest. Protein groups identified with different detection and fragmentation methods and using two or three FAIMS CVs. (B) Protein groups and unique peptides identified from single HeLa cells with and without FAIMS. Error bars indicate standard deviations based on 3 replicate measurements.

Fig. 3. Representative mass spectra obtained without (A) and with (B) FAIMS filtering (CV −55 V). Peaks corresponding to multiply charged ions in both spectra are starred.

Fig. 4. Single-cell proteomic interrogation of human spinal motor neurons and interneurons. (A) Protein groups identified from single motor neurons (MNs) and interneurons (INs). (B) Venn diagram indicating overlap of identified protein groups. (C) Principle component analysis showing differentiation of the two neuronal subtypes based on 1118 quantified features. (D) Volcano plot indicating significant differences in protein expression for quantifiable protein groups (p < 0.05, |fold difference| ≥ 2).