Proteins are wonderfully diverse! Their physicochemical diversity is essential to animating life, to performing the myriad functions necessary to process energy, nutrients and information in living systems. The physicochemical diversity of proteins also poses challenges to proteomic analysis. Different amino acid sequences can adsorb to various surfaces, thus resulting in losses, reduced sensitivity and even biases in protein sequence coverage. These problems may be especially severe with small samples, such as single cells and were among the challenges that we described in a 2018 perspective highlighting challenges and opportunities for single-cell proteomics.

Despite the potential for adsorptive losses, the degree to which such losses affect single-cell proteomics is not commonly quantified. The upper limit, corresponding to poor sample handling, is trivially 100% loss. It is more interesting to consider the lower limit: How much is lost when minute amounts of proteins and peptides are carefully handled as part of proteomic analysis?

Establishing this limit was part of a fun discussion with John Yates ||| during the first single-cell proteomics conference. Professor Yates suggested measuring adsorption losses using radioactively labeled peptides, which is a great experiment to directly measure such losses. While it has not been done recently in the context of single-cell proteomics, similar measurements have been performed by incubating about 3 billion molecules dissolved in 2ml for 48 h at 4C, or about 1 million molecules per microliter. The experiments estimated that the majority of peptides were recovered. The degree of recovery depended on the surface type, thus highlighting the importance of choosing a surface that minimizes losses.

The concentrations of peptides in these radioactive experiments were lower than those of peptides from single cells processed by a nanoliter sample preparation, such as nPOP. Thus extrapolating from the radioactive data, adsorptive losses should affect only a minority of the peptides liberated from a single cell processed in 8-20nl volumes. This is especially the case when fluorocarbon surfaces are used that should repel both hydrophilic and hydrophobic peptides. Indeed, measurements suggest that peptides from single cells are delivered to mass spectrometry detectors with about 95% of the efficiency for delivering peptides from samples composed of thousands of cells. Such losses might be further reduced by container-less cell processing. These results suggest that the fraction of proteins lost at the level of intact proteins is comparable for bulk and single-cell sample preparation but do not allow estimating this fraction in absolute terms. Absolute estimates may be derived from radioactive labeling of proteins similar to the experiments with labeled peptides.

While protein adsorption losses may not be the major bottleneck for optimized sample preparation methods, the upper limit remains 100 % loss. We need to continually evaluate, benchmark and improve the efficiency of sample handing so that adsorptive losses can be further reduced. However, the data from radioactively labeled peptides and optimized single-cell proteomics sample preparation suggest that adsorptive losses can be kept low. This is a good thing for single-cell proteomics that has many other avenues for improvement of its throughput and depth of proteome coverage.