Table 1.

Bottom-up and top-down MS-based proteomics studies on human SP.

Proteomic Approach	SP Processing and Fractionation	Samples Used for Proteomics	Normalization of Protein Quantitation	Quantification Strategies	Instrumentation/Techniques	Study Groups	References
Bottom-up	-30 min at room temperature for semen liquefaction -2000× g for 10 min at room temperature followed by 10,000× g for 20 min at 4 °C -No fractionation	58 individual samples	Yes	Label-free method (SpC 1)	1D-PAGE 2 ESI 3—Ion Trap/Orbitrap	-NZ 4 (n = 20) -AS 5 (n = 38)	Wang et al., 2009 [29]
Bottom-up	-1 h at room temperature for semen liquefaction -13,000× g for 15 min at room temperature (3 times) -RPLC 6 separation	-30 individual samples-Pool of 5 NZ samples -Pool of 5 PV 7 samples -Pool of 5 Non-obstructive AZ 8 samples	Yes	Label-free method (SpC)	ESI-Triple-Quadrupole and Ion Trap/Orbitrap	-NZ (n = 12) -PV (n = 8) -Non-obstructive AZ (n = 10)	Drabovich et al., 2011 [25]
Bottom-up	-2/3 h at room temperature for semen liquefaction -13,000× g for 10 min at room temperature -SCX 9 -RPLC separation	Pooled samples for each group	Yes	Label-free methods (SpC and XIC 10)	ESI-Ion Trap/Orbitrap	-NZ (n = 5) -Non-obstructive AZ (n = 5) -PV (n = 5)	Batruch et al., 2012 [30]
Bottom-up	-1 h (temperature not specified) for semen liquefaction -16,000 rpm for 30 min at 4 °C (3 times) -RPLC separation	119 individual samples	Yes	Stable isotope dilution SRM 11	ESI-Triple-Quadrupole	-NZ (n = 42) -Non-obstructive AZ (n = 25) -Obstructive AZ (n = 10) -PV (n = 42)	Drabovich et al., 2013 [26]
Bottom-up	-<30 min (temperature not specified) for semen liquefaction -15,000× g for 15 min at 4 °C followed by 100,000× g for 30 min at 4 °C -RP-RP HPLC 12 separation	Pooled samples for each group	Yes	Label-free method (SpC)	ESI-Ion Trap/Quadrupole/Orbitrap	-NZ (n = 11) -OAT 13 (n = 11)	Herwig et al., 2013 [31]
Bottom-up	-20/60 min (temperature not specified) for semen liquefaction -3000× g for 30 min -RP HPLC separation	Pooled samples for each group	Not mentioned	Label-free method (SpC)	ESI-Ion Trap	-NZ (n = 26) -TZ 14 (n = 22) -OZ 15 (n = 6) -OT 16 (n = 10)	Sharma et al., 2013 [28]
Top-down	-Time and temperature for semen liquefaction not specified -2000× g for 15 min followed by 14,000× g for 10 min at 4 °C -SAX 17 separation	16 individual samples	Yes	Label-free method (peak intensities)	SELDI-TOF 18 MS	-NZ (n = 7) -OA 19 (n = 9)	Cadavid et al., 2014 [37]
Bottom-up	-Time and temperature for semen liquefaction not specified -800× g (time not specified) followed by 14,000× g for 30 min at 4 °C -RP UPLC 20 separation	Pooled samples for each group	Yes	Label-free method (iBAQ 21)	ESI- Quadrupole/Orbitrap	-Adolescents without varicocele (n = 23) -Adolescent with varicocele and normal semen analysis (n = 37) -Adolescents with varicocele and altered semen analysis (n = 17)	Del Giudice et al., 2016 [27]
Bottom-up	-Time and temperature for semen liquefaction not specified -2000× g for 20 min at 4 °C followed by 10,000× g for 20 min -RP UPLC separation	17 individual samples	Yes	Label-free method	UPLC-MS	-NZ (n = 7) -AS (n = 10)	Saraswat et al., 2017 [34]
Bottom-up	-30 min (temperature not specified) for semen liquefaction -1000× g for 15 min followed by 13,000× g for 10 min (temperature not specified) -RP-RP HPLC separation	Pooled samples for each group	Yes	Label-based method (iTRAQ 22)	MALDI-TOF/TOF 23 MS	-NZ (n = 10) -OA (n = 10)	Liu et al., 2018 [35]
Bottom-up	-30 min at 37 °C for semen liquefaction -40,000× g for 30 min at 4 °C -RPLC separation	6 individual samples	Yes	Label-based (TMT 24) and label-free methods (iBAQ)	ESI-Ion Trap/Orbitrap	-NZ (n = 3) -AS (n = 3)	Wu et al., 2019 [32]
Bottom-up	-Time and temperature for semen liquefaction not specified -500× g for 10 min followed by 1500 × g for 10 min -RP HPLC separation	Pooled samples for each group	Yes	Label-based method (TMT)	ESI-Ion Trap/Orbitrap	-NZ (n = 4) -AS (n = 4) -OZ (n = 4) -AZ (n = 4)	Barrachina et al., 2019 [33]
Top-down	-15/30 min at 37 °C for semen liquefaction -15,000× g for 15 min at 4 °C -C18-bonded silica d-SPE 25	30 individual samples	Yes	Label-free method (peak intensities)	MALDI-TOF/TOF MS	-NZ (n = 15) -Infertile men (AT 26 = 2; OAT = 5; TZ = 6; OZ = 2)	Correnti et al., 2022 [36]

¹ SpC, spectral counting. ² 1D-PAGE, one dimensional-polyacrylamide gel electrophoresis. ³ ESI, electrospray ionization. ⁴ NZ, normozoospermic. ⁵ AS, asthenozoospermic. ⁶ RPLC, reversed-phase liquid chromatography. ⁷ PV, post-vasectomy (simulates obstructive AZ). ⁸ AZ, azoospermic. ⁹ SCX, strong cation exchange. ¹⁰ XIC, extracted ion chromatograms. ¹¹ SRM, selective reaction monitoring. ¹² HPLC, high performance liquid chromatography. ¹³ OAT, oligoasthenoteratozoospermic. ¹⁴ TZ, teratozoospermic. ¹⁵ OZ, oligozoospermic. ¹⁶ OT, oligoteratozoospermic. ¹⁷ SAX, strong anion exchange. ¹⁸ SELDI-TOF, surface-enhanced laser desorption/ionization- time-of-flight. ¹⁹ OA, oligoasthenozoospermic. ²⁰ UPLC, ultra-performance liquid chromatography. ²¹ iBAQ, intensity-based absolute quantification. ²² iTRAQ, isobaric tags for relative and absolute quantification. ²³ MALDI-TOF/TOF, matrix-assisted laser desorption ionization- time-of-flight/time-of-flight. ²⁴ TMT, tandem mass tag. ²⁵ d-SPE, dispersive-solid phase extraction. ²⁶ AT, asthenoteratozoospermic.