Fig. 2: MS of human sperm and parallel reaction monitoring (PRM) MS analysis of standard peptides for determination of limit-of-detection (LOD) and quantification.

a Venn diagram of identified proteins (n = 3 biological replicates) by LC-MS/MS. b Ranking of proteins according to the abundance (n = 3) (iBAQ). For abbreviations of proteins see Supplementary Tables 2–4. c Transition channels of the most sensitive PRM analysis of peptides corresponding to SCNN1A, CFTR, SLC4A4 and SLC26A6. Peptide sequences are given; asterisks denote the ¹³C- and ¹⁵N-labelled amino acids in standard peptides. Upper panels: No specific PRM signals detected for endogenous peptides derived from 90,000 sperm cells; lower panels: PRM signals detected from 1 attomole (5 attomoles for SLC26A6) of standard peptide spiked into endogenous peptides derived from 90,000 sperm cells. Different colors of the transitions signify different fragment ions. d Box plot of LOD values of standard peptides representing proteins CFTR, SLC4A4, SLC26A6, and SCNN1A monitored in n = 4 biological replicates (BioRep B1–B4) of digested sperm sample. Sequences of the standard peptides are listed. Left y-axis shows detectable amount of standard peptide in attomole spiked into endogenous peptides derived from 90,000 sperm cells. Right y-axis shows the calculated molecules/cell corresponding to the amount of detectable standard peptides. e Box plot of protein molecules/cell as determined by PRM MS for carbonic anhydrase II (CA2), chloride/anion exchanger (SLC26A3), potassium channel (Slo3), CatSper channel subunits (CatSper1, CatSper2, CatSper3, CatSper4, and CatSperB). Biological replicates are indicated by color, copy numbers were determined in n = 4 biologically independent replicates (BioRep B1–B4) of digested sperm sample from four independent experiments. For some proteins the differences between replicates were smaller than the size of the data points, in those cases less than eight data points are visible. For Slo3, only data from three of the four biological replicates could be used. Box plots represent 25%, 50% (median), and 75% quartiles, with whiskers displaying minimum and maximum data points, except for outliers outside of 1.5-times interquartile range (IQR). Source data is provided as a source data file.