The study of sperm proteins started more than a century ago with the isolation and identification by Friedrich Miescher in 1874 of a proteinaceous basic component from the sperm cell that he called “protamine” and that he found was coupled to what he called “nuclein” or what we know as DNA.13 However, it was not until about 100 years later that the protein sequencing, separation, and detection methods were developed allowing the generalized study of the proteins (Figure 18.1).14-16 Nevertheless, with these methods the proteins still had to be studied one at a time. The possibility to study the entire or a substantial proportion of the sperm proteome started much more recently, around 1995, with the application of mass spectrometry to the study of proteins (Figure 18.1).
The basic steps in most proteomic analysis at present are (1) protein or peptide extraction from the biological sample, (2) reducing the complexity of the protein or peptide extract, and (3) application of mass spectrometry and database comparisons to identify the different proteins or peptides (Figure 18.2).17 The first step as applied to the sperm cell can be accomplished either by extracting the entire sperm or fluid proteome as well as by targeting specific cell compartments such as membrane systems, nucleus, tail or organelles, or fluid components.18 The second step or reduction of the complexity of the initial protein or peptide extract can be accomplished using one-dimensional polyacrylamide gel electrophoresis (1D-PAGE) or 2D-PAGE (Figure 18.2). However, a more recent and high-throughput approach is to convert the initial protein extract into peptides on digestion with a protease and subsequently to fractionate the peptides using peptide isoelectric focusing (IEF) or monodimensional liquid chromatography (1D-LC) or 2D-LC (Figure 18.2).
The final step in a proteomic analysis is accomplished through mass spectrometry peptide and protein identification. Initial proteomic methods were developed that involved matrix-assisted laser desorption ionization—time of flight (MALDI-TOF), which relies on the accurate determination of peptide masses and comparison to peptide mass databases in search for identities. In a MALDI-TOF analysis, the proteins are typically excised from the gel, digested with trypsin, and the ratio of mass to charge of the resulting peptides determined. These peptide masses provide an accurate “peptide mass fingerprint” for
FIGURE 18.1 Pubmed publications where the keywords “protein*” or “proteome*” appear in the title. The asterisk “*” in “protein*” or “proteome*” indicates a wildcard. The year of the description of key methods to study proteins is indicated with arrows. It can be observed that proteomics is a relatively recent field as it started in 1990 with the application of mass spectrometry to study proteins.
FIGURE 18.2 Many protein analysis options are currently available. Usually sperm cells or biological material must be processed, purified, or fractionated before proceeding to extract the proteins or their targeted detection. Cells or tissue sections can be directly used using immunohistochemistry or immunocytochemistry (left). Alternatively, proteins can be separated by gel electrophoresis (center) and the desired proteins eluted and digested into peptides. A current very high- throughput approach involves the digestion of the original protein mixture by proteases (usually trypsin) to convert it to peptides (right). The final stage is to separate the peptides through liquid chromatography and to proceed to identification using mass spectrometry.
the protein and are then compared against sets of masses from databases of in silico predicted peptides derived from the genome. If several of the experimentally determined peptide masses matched with the theoretical peptide matches derived from the proteins in the databases, then it is considered that the protein has been identified.19 However, currently higher-throughput approaches based on tandem mass spectrometry (MS/MS) are being applied that also provide the opportunity for de novo peptide sequencing and posttranslational modifications detection (Figure 18.2).17
For protein quantification different possibilities are also available. Initial methods developed were based on enzyme-linked immunosorbent assay (ELISA)14 or western blot16 (Figure 18.2). These methods are extremely robust and useful but applicable only to study specific target proteins and cannot be applied to study many proteins simultaneously or even substantial proportions of the entire proteome. High-throughput approaches are currently available to quantify simultaneously many proteins in the proteome. Initial proteome quantification methods were based on measuring the protein intensities of proteins separated on 2D gels and identifying the corresponding protein spots.20,21 However, current high-throughput quantification techniques rely on peptide quantification rather than protein quantification. Peptides can be quantified by spectral counting22 or after their in vivo or in vitro labeling with tandem mass tags (Figure 18.2).23,24