Rapid Distinction of Leucine and Isoleucine in Monoclonal Antibodies Using Nanoflow. LCMS n. Discovery Attribute Sciences

Transcription

1 Rapid Distinction of Leucine and Isoleucine in Monoclonal Antibodies Using Nanoflow LCMS n Dhanashri Bagal *, Eddie Kast, Ping Cao Discovery Attribute Sciences Amgen, South San Francisco, California, United States 9 * To whom correspondence should be addressed. dbagal@ amgen.com. Phone: Fax: S-1

2 Supplemental S1: LC-MS/MS method: The tryptic peptides were introduced into the mass spectrometer using a spray voltage of 2. kv through a heated ion transfer tube at 275 C. The MS1 survey scan spectra were acquired every 3 sec at a resolution of 1, at in the Orbitrap mass analyzer over the range of 33 to 1 using data dependent acquisition (DDA). The AGC target for the MS1 scan was set to 4.E5, the maximum injection time was 5 ms and data were averaged using 1 microscan. After the survey scan, precursor ions were selected based on intensity (1.E4), dynamic exclusion criterion of sec and were subjected to subsequent fragmentation using three fragmentation techniques, namely ETD, HCD, and CID. CID uses Helium and HCD uses nitrogen as a collision gas. The NCE (normalized collisional energy) for MS 2 fragmentation was set to 35% and 28% for CID and HCD respectively. For ETD fragmentation, charge dependent calibrated parameters were used to achieve optimum ETD reaction. All the MS2 spectra were acquired in the Orbitrap mass analyzer at a resolution of 3, at, using AGC target of 5.e4, maximum injection time of ms and data were averaged using 1microscan. For all scheduled targeted MS n (tms n ) experiments, data were acquired in the ion trap mass analyzer in profile mode in order to improve sensitivity. For the w-ion method, HCD (NCE 12) was used for the MS 3 experiment and for the immonium ion method, CID (NCE 3) was used for the MS 3 experiment. Each scheduled event within an experiment was designed with a time window of ~1 min and with the following parameters: the desired ions were isolated in the quadrupole using an isolation windows of 1.6 Da and the acquisition range for the MS/MS data varied depending on the targeted experiment. Additionally for the scheduled targeted MS n events, the AGC target was set to 1.E4, the maximum injection time was set to ms and data were averaged using 2 microscans. Data analyses were S-2

3 performed using Xcaliber and Proteome Discoverer 1.4 with Sequest HT (Thermo Fisher Scientific, San Jose, CA). All the data were plotted without any processing using GraphPad Prism 6 software (GraphPad Software, LA Jolla, CA). Supplemental S2: N-Terminal Sequencing All the peptides listed in this paper were subjected to Edman degradation for amino acid sequence confirmation. N-terminal sequencing was performed on the PPSQ-33A Protein Sequencer (Shimadzu Biotechnology, Columbia, MD), where the enriched peptides were loaded onto the ProSorb PVDF membrane cartridge (Life Technologies, Grand Island, NY) and the cleaved amino acids were separated using a 4.6mm x 25mm Wakosil-PTH-II column (Wako, USA) by heating to C at a flowrate of 1mL/min. The PTH amino acids were detected using a UV absorbance at 269 nm (Data not shown). S-3

4 a Retention Time (Min) b Retention Time (Min) c Retention Time (Min) Figure S3: Extracted ion chromatograms (XIC) for (a) MS/MS of 2+ charge state ( ) for Peptide A (XSWER) generated through nano-lcms/ms using HCD (NCE 28); (b) immonium ion isolation event (, CID ) ; (c) immonium ion fragmentation event (, CID 3). S-4

5 Absolute Intensity 15 5 Absolute Intensity IM(X) Figure S4: Overlaying spectra with absolute counts for isolation (black trace) and fragmentation of immonium ion (blue trace) arising from experiments on 2+ charge state of Peptide B (SXSKPEGTPR). S-5

6 a 5 IM(X) 86. CID@ Isolation of IM (X) Absolute Intensity b Absolute Intensity CID@3 Fragmentation of IM (X) ASQNVDTYLDWYQQK Figure S5: Mass spectra of (a) isolation and (b) fragmentation of the immonium ion resulting from the Xle residue from Peptide C (ASQNVDTYXDWYQQK) plotted using absolute intensity. S-6

7 a c z c c z z z c z8 3.3 c7 * * MH z c MH-NH c z z c c z z z * 85.4 z9 z8 z z z16 Fragmentation of z8, hcd12 DSAVYYSAXLITPVVPK z ETD MS 2 DSAVYYSAXXXTPVVPK b d z Fragmentation of z7 DSAVYYcAXXITPVVPK Fragmentation of z9, hcd12 DSAVYYSAILITPVVPK z e z8 Fragmentation of z8, hcd f Fragmentation of z9, hcd z Figure S6: (a) ETD spectrum for 3+ charge state ( ) for custom synthesized Peptide S-7

8 F (DSAVYYSAILITPVVPK) generated through nano-lcms/ms (ETD reaction 5ms); (b) spectrum where the generated z7 ion is further fragmented using HCD (NCE 12); (c) spectrum where the generated z8 ion is further fragmented using HCD (NCE 12); (d) spectrum where the generated z9 ion is further fragmented using HCD (NCE 12); (e) spectrum where the generated z8 ion is further fragmented using HCD (NCE ); (f) spectrum where the generated z9 ion is further fragmented using HCD (NCE 3). (* represent the z7, z8 and z9 ions) For accurate determination of Xle residues present at the 9 th, 1 th and 11 th position in the peptide chain it is important to perform the next level of mild HCD fragmentation on the corresponding z9, z8, z7 ions. Figure S5-b shows that mild HCD fragmentation (NCE 12) of the z7 ion ( 737.5) produces a w-ion at 78.3 which is 29 Da lesser than the precursor ion. This loss of 29 Da indicates that the residue presents at the 11 th position from the N-terminus in the peptide chain is indeed Ile. Similarly HCD fragmentation of z8 ( 85.4) and z9 ( 963.5) ions shown in Figure S5-c and S5-d produce w-ions that are 43 Da and 29 Da lesser than the precursor ions respectively. These respective losses of 43 Da and 29 Da indicate the presence of Leu residue in the 9 th and that of Ile residue in the 1 th position in the peptide chain. The resulting spectra are clean, demonstrating that radical site migration effect is not observed at such low HCD voltages. However increasing the HCD voltage results in radical site migration. Figure S5-e shows that use of HCD (NCE ) results in an additional peak at which is 29 Da lesser than the precursor z8 ion. This loss of ethyl group arises from radical site migration effects since the amino acid adjacent to the terminal Leu containing z8 ion is Ile. S-8

9 Similarly figure S5-f shows that the use of HCD (NCE3) results in an additional peak at 9.5 which is 43 Da less than the precursor z9 ion. This loss of isopropyl radical arises from radical site migration since the amino acid adjacent to the terminal Ile containing z9 ion is Leu. Peak at results from loss of methyl group (-15 Da) which is sometimes observed as a result of Ile fragmentation. S-9