Mammalian cells have about 25,000-fold more protein molecules than mRNA molecules. This larger number of molecules and the associated larger dynamic range have major implications in the application of proteomics technologies. We examine these implications for both liquid chromatography-tandem mass-spectrometry (LC-MS/MS) and single-molecule counting and provide estimates on how many molecules are routinely measured in proteomics experiments by LC-MS/MS. We review strategies that have been helpful for counting billions of protein molecules by LC-MS/MS and suggest that these strategies can benefit single-molecule methods, especially in mitigating the challenges of the wide dynamic range of the proteome. We also examine the theoretical possibilities for scaling up single-molecule and mass-spectrometry proteomics approaches to quantifying the billions of protein molecules that make up the proteomes of our cells.

We analyzed this sample with a Thermo EASY-nLC 1200 coupled with a Thermo Orbitrap Eclipse Tribrid Mass Spectrometer. We used a 30 cm fused silica Picofrit (New Objective) 75 µm column and 3.5 cm 150 µm fused silica Kasil1 (PQ Corporation) frit trap loaded with 3 µm Reprosil-Pur C18 (Dr. Maisch) reverse-phase resin Solvent A was 0.1% formic acid in water, and solvent B was 0.1% formic acid in 80% acetonitrile. For each 3 µl injection, we loaded ∼1.5 μg peptides and separated them using a 90-min gradient (0 to 40% B over 80 min, 40 to 75% B over 10 min), followed by a 10 min wash (100% B). The MS spray voltage was 2 kV with a capillary temperature of 300 °C. For the 6 chromatogram library acquisitions, we used 4 m/z precursor isolation windows at 30,000 resolution (AGC target 1000% or 5e5, Auto maximum inject time, 27 NCE). The spectra were acquired using a staggered window pattern with window placements optimized by Skyline (i.e., 398.43–502.48, 498.48–602.52, 598.52–702.57, 698.57–802.61, 798.61–902.66, and 898.66–1002.70 m/z). A precursor spectrum matching the range (i.e., 395–505, 495–605, 595–705, 695–805, 795–905, and 895–1005 m/z) using a Standard AGC target and a maximum inject time of 50 ms were interspersed every 25 MS/MS spectra. For the single-injection run, we acquired 75 x 8 m/z (400–1000 m/z) precursor isolation window DIA spectra (15,000 resolution, AGC target 1000% or 5e5, Auto maximum inject time, 27 NCE) using a staggered window pattern with window placements optimized by Skyline. Precursor spectra (target range ± 5 m/z at 30,000 resolution, Standard AGC target, Auto maximum inject time) were interspersed every 75 MS/MS spectra.

Extracellular vesicles (EV) were enriched from 1.75 mL banked plasma using the Qiagen ExoEasy Maxi kit (Qiagen) according to manufacturer’s instructions. The protein concentration of the resulting EV sample was determined using Pierce BCA Protein Assay Kit (ThermoFisher Scientific) according to manufacturer’s instructions. The EV sample (50 µg) was combined with enolase (800 ng) and reduced with TCEP (10 mM final concentration) for 1 hr at 37C, alkylated with IAA (15 mM final concentration) for 30 min in the dark at RT, and quenched with DTT for 15 min at RT. The reduced sample was then precipitated and digested with trypsin using the SP3 automated method in the Kingfisher robot. The eluted peptide sample was quenched with the addition of TFA to a final concentration of 0.5%. PRTC was added to the peptide sample to 50 nmol/µl final concentration.