Oocytes undergo a range of complex processes via oogenesis, maturation, 
fertilization, and early embryonic development, eventually giving rise to a 
fully functioning organism. To understand proteome composition and diversity 
during maturation of human oocytes, here we have addressed crucial aspects of 
oocyte collection and proteome analysis, resulting in the first proteome and 
secretome maps of human oocytes. Starting from 100 oocytes collected via a novel 
serum-free hanging drop culture system, we identified 2,154 proteins, whose 
function indicate that oocytes are largely resting cells with a proteome that is 
tailored for homeostasis, cellular attachment, and interaction with its 
environment via secretory factors. In addition, we have identified 158 
oocyte-enriched proteins (such as ECAT1, PIWIL3, NLRP7)(1) not observed in 
high-coverage proteomics studies of other human cell lines or tissues. 
Exploiting SP3, a novel technology for proteomic sample preparation using 
magnetic beads, we scaled down proteome analysis to single cells. Despite the 
low protein content of only ∼100 ng per cell, we consistently identified ∼450 
proteins from individual oocytes. When comparing individual oocytes at the 
germinal vesicle (GV) and metaphase II (MII) stage, we found that the Tudor and 
KH domain-containing protein (TDRKH) is preferentially expressed in immature 
oocytes, while Wee2, PCNA, and DNMT1 were enriched in mature cells, collectively 
indicating that maintenance of genome integrity is crucial during oocyte 
maturation. This study demonstrates that an innovative proteomics workflow 
facilitates analysis of single human oocytes to investigate human oocyte biology 
and preimplantation development. The approach presented here paves the way for 
quantitative proteomics in other quantity-limited tissues and cell types. Data 
associated with this study are available via ProteomeXchange with identifier 
PXD004142.