New Methods For Mass Spectrometry-based Proteomics Analysis Using Mass-limited Samples

Proteins are the core of life activities,and play a crucial role in maintaining normal metabolism and substance transport in the body.With the rapid development of mass spectrometry,it has become an important tool for gaining deeper understanding of the functions and roles of proteins.Mass spectrometry-based proteomics methods is able to quantify thousands of proteins in complex biological samples.Protein requires multiple steps of processing before analyzed by mass spectrometry.This process results in a significant sample loss.In order to achieve deep coverage,typically,proteomics analysis requires >1μg samples for deep coverage.Sample preparation is a key step,and the quality of samples can directly affect the results of analysis and identification.However.It was difficult to obtain a sufficient sample for the study of rare cells such as germ cells and circulating tumor cells.From the perspective of sensitivity,the detection of proteins in small amounts/single cells is challenging.Therefore,it was urgent to develop efficient sample preparation methods to reduce protein/peptide loss for in-depth proteomics analysis of rare cell samples.Many methods have been developed for the study of trace sample.Nowadays,the popular strategy is to minimize the reaction system volume.This strategy usually requires specialized equipment or trained personnel to operate,which is difficult to apply in ordinary laboratories,and therefore limiting their wide application.Here,we report an angled-shape tip-based strategy for rapid sample preparation and sensitive proteomic profiling of small cell populations(<1000 cells).The angled-shape tip provided a ‘reactor’ for the entire proteomic sample processing workflow,from cell capture and lysis to protein digestion,eliminating the sample transfer-induced protein loss.The angled-shape tip was surface-treated for anti-protein adsorption and further reduced the sample loss.Using this strategy,1241±38～4110±37 protein groups and4010±700～34879±575 peptides were identified from 10-1000 He La cells with high quantification reproducibility using only 4.5 h sample processing time,which was superior to the reported methods and commercial kits,especially for <100 cells.This approach was compatible with flow cytometry-based cell sorting,easily accessible and straightforward to operate in a routine laboratory.For the angled-shape tip strategy,the sample volume was still too large for single cell sample processing.Therefore,we established a sample preparation platform by combining flow cytometry and micro-chip with volume of only 600 n L as the reaction vessel.This platform had high throughput cell sorting using flow cytometry sorter,which could separate different subtypes and different numbers of cells quickly and accurately.Furthermore,the small surface area of micro-chip results in reduced nonspecific adsorption,and higher enzymatic digestion efficiency for trace sample.Using this platform,987±10 to 3120±17 proteins and 5540±114 to 20738±428 peptides were identified from 1-100 He La cells.About this 2500 proteins and 15000 peptides were identified by applying strategy to 100 mouse hepatocytes.Further analysis of these protein groups explored the changes in the mouse liver hepatocyte proteome under different culture conditions,providing a foundation for studying primary cells using flow-based microfluidic single-cell sample preparation platforms.