A proper internal standard choice is critical for accurate, precise, and reproducible mass spectrometry-based proteomics assays. Synthetic isotopically labeled (SIL) proteins are currently considered the gold standard. However, they are costly and challenging to obtain. An alternative approach uses SIL peptides or SIL "winged" peptides extended at C- or/and N-terminus with an amino acid sequence or a tag cleaved during enzymatic proteolysis. However, a consensus on the design of a winged peptide for absolute quantification is missing. In this study, we used human serum albumin as a model system to compare the quantitative performance of reference SIL protein with four different designs of SIL winged peptides: i) commercially available SIL peptides with a proprietary trypsin cleavable tag at C-terminus, ii) SIL peptides extended with five amino acid residues at C-terminus, iii) SIL peptides extended with three and iv) with five amino acid residues at both C- and N-termini. Our results demonstrate properties of various SIL extended peptides designs, e.g., water solubility and efficiency of trypsin enzymatic cleavage with primary influence on quantitative performance. SIL winged peptides extended with three amino acids at both C- and N- termini demonstrated optimal quantitative performance, equivalent to the SIL protein.

HSA concentration in the pooled serum sample diluted 2-fold with ultrapure water was determined using BCG albumin assay kit on the 96-well plate format (cat. #MAK124-1KT, Sigma-Aldrich). The seven-point calibration series was prepared using HSA standard (5 to 50 mg/mL) in ultrapure water. The absorption spectrophotometry was measured at 620 nm wavelength. The pooled serum sample (10 µL), diluted 2000-fold using 50 mM AmBic with 5 mg/mL SDC (AmBic/SDC buffer) to HSA concentration of approx. 500 nM and transferred into a clean microcentrifuge tube. After adding SIL protein or peptide internal standard (10 µL), the sample was reduced (adding 1 µL of 200 mM DTT stock solution, 95°C, 10 min), cooled down to ambient temperature, and cysteine residues were alkylated (adding 1 µL of 400 mM IAAstock solution, ambient temperature, 30 min in the dark). Trypsin was added to the sample in a 1:20 ratio (w/w enzyme to total protein content), and samples were incubated (37°C, 16 h) in microcentrifuge tubes sealed with a paraffin film. The enzymatic proteolysis was quenched at different time points (0.5, 2, 4, 8, 16, and 24 h; n=3) to optimize the incubation time. The proteolysis was quenched by adding 200 µL of 2% FA in water (pH < 3), and tryptic peptides were desalted using a mixed-mode SPE cartridge in a 96-well plate format (Oasis PRIME HLB, Waters, Milford, MA). Samples were loaded onto the SPE cartridge, washed (300 µL water with 2% FA, pH < 3), eluted (50% ACN; 2% FA in water, pH < 3), and the extract was dried in a vacuum concentrator centrifuge (Savant SPD121 P SpeedVac, Thermo Fisher). Samples were dissolved (10 µL) in 5% ACN with 0.1% FA in water before UHPLC-SRM analysis. Liquid chromatography and mass spectrometry protein assays. Samples were analyzed using the UHPLC system (InfinityTM 1260 Agilent Technologies, Santa Clara, CA) and a reversed-phase analytical column (C18 Peptide CSH; 1.7 µm, 2.1 mm i.d. x 100 mm; cat. #186006937; Waters; Milford, MA). The column and the autosampler temperatures were 40°C and 8°C, respectively, and the sample injection volume was 3 µL. The mobile phase flow rate was 0.3 mL/min using mobile phase A (0.1% FA in water) and B (0.1% FA in 95% ACN) in the linear gradient elution mode (0-15 min) with a wash step (15.30-20 min) and re-equilibration step (21-25 min). The gradient program was: 0.0 min 5% B; 15 min 20% B; 15.3 min 95% B; 20 min 95% B; 21 min 5% B; 25 min 5% B. A triple quadrupole mass spectrometer (6495B, Agilent Technologies, USA) was used for SRM assays in positive ion mode. A standard-flow Jet Stream electrospray ionization (ESI) source parameters were: capillary voltage 3.5 kV, gas flow rate 18 L/min at 220°C, sheath gas pressure 25 PSI and flow rate 12 L/min at 400°C and nozzle voltage 800 V. The acquisition in dynamic SRM mode was centered on the peptide experimental retention time within 3 min window. All tryptic peptides and SIL peptides internal standards were analyzed using 3-5 SRM qualifiers and one quantifier SRM transition. In total, 96 transitions were monitored with a total cycle time of 500 ms.

Individual human serum samples were collected during a one-time morning session from 14 healthy adult volunteers (seven women and seven men). Venous blood collected into 9 mL serum tubes was allowed to clot and centrifuged (10 min, 2,500 g, 20°C). Individual serum samples were pooled and stored at 20°C until analysis. Signed informed consent forms were obtained from all participants and archived. The study was approved by the Committee for Ethics of CELSPAC: TNG (CELSPAC/EK/4/2016) at University Hospital Brno, Czech Republic, under the Declaration of Helsinki.