Research Article Yang, Kernstock, Simmons & Alak
cleavage. After cooling and centrifugation, alkylation was performed by adding 30 μl of 50 mmol/l IAA pre- pared in Buffer C and incubating for 60 min at room temperature with light protection. The digestion was conducted by adding 10 μl of 0.1 μg/μl trypsin and incubating 4 h at 37°C. The reaction was stopped by adding 10 μl of 5% formic acid.
LC–MS/MS instrumentation & conditions Qualitative and quantitative experiments were per- formed using a Shimadzu system comprised of LC- 20AD XR HPLC pumps and SIL-20AC XR autos- ampler coupled with an AB SCIEX 5500 QTrap mass spectrometer. Separation was achieved on an Aeris Peptide XB-C18 analytical column (150 × 2.1 mm, 3.6 μm) with a UHPLC C18-Peptide Security Guard Cartridge (Phenomenex), using the mobile phases con- sisting of (A) 0.1% formic acid in water and (B) 0.1% formic acid in ACN. Elution was programmed by increasing mobile phase B from 2% at 0 min to 40% at 10 min with a flow rate of 0.35 ml/min. The digested tryptic peptide solution (5 μl), stored at 10°C in the autosampler, was injected into the system. The mass spectrometer was operated in positive ESI
mode. The system was extensively optimized using Instrument Optimization mode and Compound Opti- mization mode through infusing tryptic peptide solu- tion of the standard purified by C-18 cartridges and the IS solution. The optimized conditions for quanti- tation were as follows: ionspray voltage 5500 V, source temperature 550°C, GS1 30, GS2 20, curtain gas 10, CAD gas medium, EP 10, CE 35, CXP 22, DP 51, dwell time 150 ms, unit resolution for both Q1 and Q3, multiple reaction monitoring (MRM) mass tran- sition for analyte surrogate peptide m/z 772.6 (+2) → 969.5 and MRM mass transition for IS m/z 775.6 (+2) → 975.5.
Results & discussion LC–MS/MS method development The analyte, ASP2409, used for evaluating the micro- plates and magnetic beads is a therapeutic protein being developed at Astellas. It is a fusion protein of mutated CTLA-4 fused to the Fc fragment pf human immuno- globin, with a dimeric molecular weight over 75 KDa. Generally, MS-based quantitation approaches could be performed either with intact protein or with its pep- tide representatives, that is, surrogate peptides. The direct approach is always preferred whenever possible for peptides and smaller proteins (typically <10 kDa) using low resolution MS instruments, and for larger proteins using high resolution and high accurate MS systems because it eliminates the inherent variability introduced by enzymatic digestion [18]. However, the
310 Bioanalysis (2015) 7(3)
approach has several limitations. It requires a high res- olution and high mass accuracy system. Proteins could be present in multiple charged ion states when ionized by electrospray. This ion distribution could dilute the signal intensity, resulting in reduced detection sensi- tivity. In this work, the second approach was selected to maximize sensitivity for this protein using an AB SCIEX 5500 Qtrap system, which has an upper m/z limit of 1250. In silico digestion of the protein yields 30 tryptic
peptides; 24 of which possess m/z (+2) within the detection window (≤1250). After Swissprot data- base search, 21 peptides were excluded because they overlapped with other endogenous human proteins. Among the three remaining candidates, a peptide with the sequence GIASFVCEYESPGK was selected as the surrogate peptide because it exhibited good chromato- graphic behavior and ESI response. This surrogate peptide contains 14 amino acids yielding a precursor ion of m/z 772.4 (+2). Its y7 ion (m/z 969.4 (+1)) was a dominant product ion and was selected as a frag- ment for MRM monitoring. After tryptic digestion, the synthesized internal standard generated a peptide with the sequence GIASFVCEYESP (13
MRM mass transition m/z 775.6 (+2) → 975.5 was used for quantitation. The analytical system was fully optimized for sensitivity using C18 cartridge- purified tryptic digestion of the reference standards, and was further optimized for selectivity and carryover using human serum samples subjected to immunoaffinity purification and tryptic digestion.
C5,15
Conditions for immobilization of capture protein The mechanism of immobilizing a capture protein on a solid surface can be either passive adsorption or chemical binding, depending on the surface proper- ties, and immobilization efficiency is influenced by many conditions such as protein concentration, buffer salt and pH, incubation time and temperature. Opti- mizing these factors involved incubating 150 μl CD86 solution under various conditions with the surfaces, followed by using the coated surfaces to capture 200 μl of 1 μg/ml ASP2409 in human serum, digesting the captured ASP2409 on the surfaces and analyzing the surrogate peptide by LC–MS/MS. MA plates are surface bonded with maleic anhydride.
Protein immobilization to the surface is through nucleo- philic addition of amine moieties in proteins with elec- trophilic anhydride groups attached on the surface. The immobilization on Tosylactivated magnetic beads and NIA surface exhibits the same chemistry, but the elec- trophilic group on the beads is tosyl chloride, while the active groups on NIA surface have not been revealed.
future science group N)GK. Its
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