While such work highlights the utility of comparative proteomic strategies as a powerful starting point for dissecting the functional lesions associated with male infertility, the main disadvantages of this approach are concerned with post-experimental data processing. Indeed, the extremely large volume of data collected in such experiments presents a significant problem in terms of both the time required to collate and assemble the data into a useable format and the computing power needed to complete database searching. This problem may eventually be alleviated as mass spectrometric instrumentation, sequencing algorithms, and the performance of computing resources continue to improve and become more affordable. These improvements, coupled with the increasing stringency of data requirements of journals are helping to make results more transparent and address the burden of erroneous identification of proteins that appear in many published proteomes [90].

SRM is an absolute quantitation method that exploits the unique capabilities of a triple quadrupole mass spectrometer. This analysis is performed by the acquisition of selected events across the LC retention time domain of predefined pairs of precursor and product ion masses. The technique becomes an absolute quantitation tool by spiking isotope-labeled synthetic peptide(s) into the complex sample of interest, which acts as an internal standard for any peptide(s) of interest. The labeled peptide standards are designed to mimic those generated by tryptic sample digestion, thus enabling them to co-elute and be subjected to MS/MS analysis along with the target peptides. A calibration-response curve based on the labeled peptides is subsequently used to accurately determine the absolute concentration of targeted peptides, a procedure that is repeated for each target within the sample. It follows that assay development and optimization are key elements of the SRM proteomic strategy. Indeed, the labeled peptides must be synthesized based on a priori knowledge for each target, taking into account those tryptic fragments that possess optimal electrochemical characteristics. While sophisticated software packages are available to help predict the most suitable peptide sequences, an element of trial-and-error and the necessity for instrument optimization renders the process of quantitation via SRM a time-consuming and costly endeavor. Notwithstanding such limitations, SRM is now firmly established as a method of choice for quantitative clinical applications. This reflects its unparalleled ability to characterize and quantify a set of proteins reproducibly, selectivity and with high sensitivity. Indeed, SRM readily extends analytical capability to low-abundance proteins without bias from abundant analytes, with recent reports suggesting the technique can detect proteins with as few as 50 copies per cell from among complex unfractionated lysates [91].

In one of the first applications to illustrate the potential of SRM in the context of male infertility research, Drabovich et al. employed the technique to assess a cohort of prospective seminal plasma biomarkers for their ability to discriminate between fertile, post-vasectomy and non-obstructive azoospermia patients [92]. From an initial investigation of 31 proteins identified in a multiplex SRM assay, the authors synthesized heavy isotope-labeled internal standards to reanalyze the concentration of 20 of the most promising candidates [92]. This approach was subsequently extended through investigation of diagnostic biomarkers to differentiate between obstructive and non-obstructive azoospermia [93]. In both instances, key biomarkers were identified that performed with either absolute, or nearly absolute, specificities and sensitivities in these assays. Such results offer the promise of developing viable alternatives to alleviate the current need for invasive testicular biopsy as the only definitive diagnostic method to distinguish between obstructive and non-obstructive azoospermia.

The recent advent of faster acquisition MS equipment has fueled the development of a new proteomic approach referred to as SWATH-MS. In essence, SWATH-MS allows the generation of a complete and permanent spectral library constituting a record of all fragment ions of the peptide precursors present in a biological sample. In combining the unique and material advantages of traditional shotgun (high throughput) and SRM (high reproducibility and consistency) technologies, SWATH-MS can be deployed for both discovery and quantitation of all detectable peptides present in complex biological samples. It also affords the added advantage that it does not rely on prior knowledge of the precursor peptide ions, instead acquiring information in a data-independent manner and thus avoiding laborious assay development. The SWATH-MS workflow involves two key steps beginning with the generation of a spectral library (e.g. via conventional LC-MS/MS) through which acquired peptides are identified. During this acquisition mode, the mass spectrometer is programed to step within 2–4 s cycles through a set of precursor acquisition windows covering the mass range accessible by a quadrupole mass analyzer and also that in which most tryptic peptide precursors should fall (400–1200 m/z). During each cycle, the mass spectrometer fragments peptide precursors and records a complete, high accuracy fragment ion spectrum for all precursors that elute on the chromatograph. This is then followed by acquisition of SWATH-MS data for each sample under analysis, interrogating and matching against the spectral library to identify peptides, and finally extraction of specific peptide ions to enable area-under-the-curve quantitation between samples.