Liquid chromatography – mass spectrometry

3.9.1 Nano-LC – ESI Q-Tof mass spectrometry

LC/MS analyses were performed using a Waters Q-Tof Premier mass spectrometer equipped with a nanoAcquity UPLC system (Waters, Milford, MA). Analyses were performed on a 75 µm × 100 mm, Atlantis 3 µm dC18 column (Waters, nanoAcquity), using a flow rate of 300 nL/min. A C18 trapping column (180 µm × 20 mm) with a 5 µm particle size (Waters, nanoAcquity) was positioned in-line with the analytical column and upstream of a micro-tee union used both as a vent for trapping and as a liquid junction.

Trapping was performed for 3 minutes at a 5 µL/min flow rate, using the initial solvent composition. Briefly, a 4 µl aliquot of the digest sample was injected onto the column.

Peptides were eluted using a linear gradient from 98 % solvent A (0.1 % formic acid in water (v/v)) and 2 % solvent B (0.1 % formic acid in acetonitrile (v/v)) to 40 % solvent B over 60 minutes. For some experiments this gradient was extended over a 90 minutes time window. Mass spectrometer settings for the MS analyses were: capillary voltage, 3.2 kV; cone voltage, 33 V; collision energy, 8.0 V; and source temperature, 80^○ C. The mass spectra were acquired over the mass range 200 – 2000 Da.

MS/MS data were acquired in the data dependent mode, using collision energies based on mass and charge state of the candidate ions. Alternatively, collision energy ramps from 20 V to 30 V, and from 30 V to 40 V, respectively, were employed in order to obtain optimal fragmentation of the (glyco)peptide ions. Representative examples of MS/MS data with the complete bx-yz nomenclature [182-184], obtained for the N-terminal anti-Aß(1-17) antibody heavy chain are shown in Figure 3.2, Figure 3.3, Figure 3.4 and Figure 3.5. A capillary voltage of 3.2 kV and a cone voltage of 20 V were used for glycopeptide analysis, in order to prevent their in-source decomposition. For subclass-specific glycosylation analysis, the instrument was operated in the MS only mode; for each sample, technical triplicates were

acquired. An external lock mass using a separate reference sprayer (LockSpray) and a solution of Glu-Fibrinogen peptide (300 fmol/µL) in water/acetonitrile 80:20 (v/v) and 0.1 % formic acid, with a mass of 785.8496 (2+), was used for calibration. Data analyses were performed using using MassLynx 4.0 software (Waters, Milford, MA).

Figure 3.2: Collision induced dissociation spectrum of the precursor ion of m/z 984.54 (2+) corresponding to the N-terminal region VH(1-19) of the 6E10 antibody heavy chain, indicated at the top. The peptide was assigned by NCBInr protein database search. The observed amide bond cleavages are indicated in the peptide sequence. The asterisk (*) indicates loss of ammonia from the y fragment ions.

Figure 3.3: Fragment ion spectrum (CID) of the precursor ion of m/z 747.38 (3+) assigned to peptide VH (20-38) of the 6E10 heavy chain. The sequence of the peptide VH(1-19) and the observed backbone cleavages are indicated at the top. The spectrum was obtained on the Q-Tof Premier using data dependent acquisition, with a collision energy ramp from 30 V to 40 V.

Figure 3.4: Fragment ion spectrum of the precursor ion of m/z 490.26 (3+) assigned by NCBInr database search to the peptide VH(39-50) of the 6E10 antibody heavy chain. The peptide sequence and the observed amide bond cleavages are indicated at the top (right). The asterisk (*) indicates loss of ammonia from the b and y fragments during collision induced dissociation.

Figure 3.5: Fragment ion spectrum of the precursor ion of m/z 864.96, corresponding to peptide VH(105-120) from the 6E10 antibody heavy chain. The peptide sequence and observed backbone cleavages are indicated at the top. The loss of water from the b and y fragment ions arising during collision induced dissociation is indicated with circles (○). The spectrum was obtained on the Q-Tof Premier using data dependent acquisition, with a collision energy ramp from 30 V to 40 V.

3.9.2 Nano LC – ESI Ion trap mass spectrometry

Nano-LC/ESI/MS/MS analyses were performed using an Agilent 6340 (Santa Clara, CA) Ion Trap equipped with an HPLC Chip Cube MS interface, an Agilent 1200 nanoHPLC and an electron transfer dissociation module. The solvents used for chromatography were 0.1% formic acid in deionized water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Ion trap-MS/MS analyses were performed as follows: 20 µL injections of the tryptic or chymotryptic digests dissolved in 0.1% formic acid were loaded onto a 40 nL enrichment column followed by a 43 mm x 75 µm analytical column, packed with ZORBAX 300SB C18 particles. Linear gradients of 3-50% solvent B were performed over 50 min at a flow rate of 500 nL/min. The parameter settings for positive ion ESI-MS were as follows:

capillary voltage – 2000 V, end plate offset – 500 V, capillary exit – 180 V, nebulizer – 2 psi, dry gas – 4 L/min, dry gas temperature – 325 °C. For MS/MS (ETD or CID), automated data dependent acquisitions of the four most abundant ions were employed.

For CID, the fragmentation amplitude was 0.80 V, which was scanned from 30% to 200%

of this preset value (SmartFrag parameter on the instrument tune page). For ETD

analyses, the accumulation time of gaseous fluoranthene anions was 10 – 12 msec, and the reaction time was typically 100 or 150 ms. The ETD/CID voltage was set at 0.07 – 0.10 V when supplemental ion activation of the charged reduced molecule radical ions was employed.

3.9.3 Analytical RP – HPLC combined with "off-line" ESI – Ion trap MS

3.9.3.1 RP-HPLC

Separation of the Aß-autoantibody heavy and light chain tryptic digests was performed on a Bio-Rad analytical HPLC system (Bio-Rad, Richmond, VA) equipped with a 4.6 mm × 250 mm Vydac 5 µm C4 column, using a flow rate of 0.5 mL/min. The mobile phases were:

0.1 % trifluoroacetic acid (TFA) in deionized water (v/v, solvent A), and 80 % acetonitrile in deionized water (v/v), acidified with 0.1 % TFA (v/v, solvent B). A linear gradient ranging from 0 to 65 % solvent B over 130 minutes was employed for peptide separation. The UV detection of the eluting peptides was performed at 220 nm, representing the absorption wavelength characteristic for the amide bond. The eluting fractions were collected in 1 mL Eppendorf tubes and lyophilized to dryness.

3.9.3.2 "Off-line" ESI – ion trap mass spectrometry

Prior to MS, the lyophilized peptide fractions were resuspended in 12-15 µL 0.1 % formic acid in deionized water (v/v). LC-ESI-Ion trap MS was performed on an Esquire 3000 ion trap instrument (Bruker Daltonik, Bremen, Germany) connected to an Agilent 1100 HPLC system (Santa Clara, CA). LC-analyses of individual fractions were performed on a 1 mm

×10 cm Discovery BIO Wide Pore 3 µm C18 column (Sigma-Aldrich, St. Louis, MO), at a flow rate of 50 µL/min. The mobile phases were: 0.1 % formic acid in deionized water (v/v, solvent A) and 80 % acetonitrile in deionized water (v/v), acidified with 0.1 % formic acid (v/v, solvent B). A linear gradient from 20 to 50 % to solvent B over 3 minutes was employed for peptide separation. For each fraction, 4-5 injection cycles (3 µL injection/cycle) were performed, as follows: (i) a first analysis was performed using MS only acquisition, in which potential multiply charged peptide precursor ions were identified, and (ii) subsequent injection cycles were performed using targeted MS/MS acquisition of the precursor ions observed in the first injection cycle; a typical value of the fragmentation

amplitude employed to achieve dissociation of the precursor ions was 1 V. Additional MS parameters were: capillary voltage – 3000 V, capillary exit – 120 V, skimmer – 40 V, dry gas temperature – 300 V, dry gas – 7 L/ min, nebulizer gas pressure 14-16 psi. The total analysis time for one injection cycle was 25 minutes. Singly charged ions were not considered for targeted MS/MS.