Sponsored by Thermo.

Attendance is free of charge, compliments of our sponsor!

Please register here.

Featured Speakers

I. Jonathan Josephs, Ph.D

Principal Scientist, Bristol-Myers Squibb

“Creation of Rich MSn Data for Structure Elucidation in Quan/Qual Analysis: Applications of Combined HCD/CID and New Isolation Techniques”

II. Dr. Jie Qian

TBD TBDn

“Orbitrap Analysis of Native Intact Monoclonal Antibody”

Program

5:30 pm Social and registration
6:15 pm Complimentary dinner
7:00 pm Welcome and opening remarks
7:05 pm Jonathan Josephs
8:00 pm Jie Qian
8:55 pm Closing remarks

Abstract for Talk II:

Introduction — Because monoclonal antibodies (mAbs) are increasingly an important line of therapeutics for the biopharmaceutical industry, the demand to better understand the biochemical and biophysical properties of mAbs has become critical. Recent developments in native protein mass spectrometry (MS) have clearly shown its distinctive power for characterization of intact proteins and protein conjugates. The mass spectrometry–based study of intact mAbs in native-like states provides a wealth of information to interpret its biological features. This is the first reported use of a commercial Orbitrap mass analyzer for measuring a native intact mAb protein. A monoclonal IgG1 kappa antibody, composed of two heavy chains of 451 amino acids and two light chains of 213 amino acids was characterized in this study.

Methods — The mAb was analyzed using normal flow liquid chromatography (LC)-MS method. The intact mAb protein was eluted from a size exclusion column under non-denaturing and non-reducing conditions and then directly detected by a standard stock bench-top Orbitrap mass spectrometer under full scan MS mode. The AGC target was set at 3e6 with resolution at 17,500. The MS spectrum was then analyzed with protein deconvolution software (Protein Deconvolution 2.0) that utilizes the ReSpect algorithm for molecular mass determination.

Results — To monitor the IgG1 kappa in native-like state, the protein was dissolved under non-denatured and non-reduced conditions. The protein was then chromatographically eluted from a size exclusion column and directly analyzed online using an Orbitrap mass spectrometer with high mass range. The mass spectra from LC-MS analyses of the native intact antibody demonstrate a clean distribution of the multiply charged envelope at mass range above 5000 m/z. Individual charge states in the raw mass spectrum for the native antibody shows major glycosylation species. Results from this study demonstrate an efficient and potentially high through-put workflow for antibody measurement in native-like states. This protocol, using a combination of normal flow size exclusion LC and a HR/AM bench-top Orbitrap mass spectrometer is expected to provide important data for mAb studies.

Novel Aspect — Native Intact Monoclonal Antibody was analyzed by a standard stock bench-top Orbitrap mass spectrometer

Past MSDG Events

This meeting is sponsored by

Featured Speakers

Talk I
19F NMR as a Direct Probe of Intermolecular Interactions by Non-Structural Protein 1 from Influenza A Virus

James M. Aramini, PhD,

Research Assistant Professor, Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ

Talk II
“Nexomics Biosciences”

Gregory Kornhaber, PhD,
Nexomics Biosciences, North Brunswick, NJ

Abstract for Talk I:

Non-structural protein 1 of influenza A virus, NS1A, is a key multifunctional virulence factor produced in the infected host cell that plays a critical role in evading the host antiviral response. This highly conserved hub protein in influenza infection is comprised of two domains: an N-terminal double-stranded RNA-binding domain (RBD) and a C-terminal effector domain (ED) which binds to a plethora of host cellular proteins. Isolated RBD and ED domains of NS1A both exist as homodimers in solution.

Since the 1960s, 19F has been recognized as a valuable NMR probe for biological systems due to its numerous favorable NMR properties, including its nuclear spin (I = ½), high natural abundance (100%), extremely high sensitivity, wide chemical shift range, minimal inherent background 19F signals, and the exquisite sensitivity of its chemical shift to changes in local environment.

Here we have applied 19F NMR to the study of NS1A from influenza A/Udorn/307/1972 (H3N2) virus. We incorporated 5-fluorotryptophan (5-F-Trp) into NS1A RBD, NS1A ED, and full-length NS1A, and assigned the 19F signals corresponding to the four Trp residues in this protein by site directed mutagenesis. We demonstrate that the 19F signal for W187, located in a functionally important helix-helix interface, can be used to monitor the oligomerization state of the ED (i.e., dimer to monomer transitions). Our 19F NMR data provide strong evidence in favor of conformational exchange at this critical interface in solution. Moreover, for full-length NS1A, 19F NMR provides the first direct spectroscopic evidence that W187, which is known to mediate intermolecular ED:ED interactions in the oligomerization of full-length NS1A, becomes solvent-exposed at protein concentrations below aggregation (ca. 50 %mu;M). Finally, we demonstrate that 5-F-Trp and 4-fluorophenylalanine (4-F-Phe) incorporation combined with 19F NMR serves as a sensitive approach for monitoring the binding of the F2F3 peptide from CPSF30 to NS1A ED.

This work was supported by grants from the NIGMS Protein Structure Initiative U54-GM094597 (to G.T.M.), NIH U01-AI074497 (to G.T.M. and R.M.K.), and NIH R01-AI11772 (to R.M.K.).

The Mid-Atlantic Lab Robotics Interest Group meets several times a year.
Details at their own website: www.lab-robotics.org.

The Teacher Affiliates (TA) are a group of high school teachers from North Jersey who are affiliated members of the Section.

The Executive Committee of the group meets monthly. Details and dates can be found at the group’s website.

The TA group sponsors or co-sponsors several activities throughout the year including the New Jersey Science Convention in October, Chemistry Day and Week activities in October, Professional Development Workshops, and the New Jersey Chemistry Olympics in May.

Attendance is free of charge, compliments of our sponsors!

Please register here.

Featured Speakers

I. Xinping Fang, Ph.D

VP, Head of Bioanalytical & Acting Head of Drug Metabolism / Biotransformation, XenoBiotic Laboratories, Inc, Plainsboro, NJ.

“Implementing an Ultrasensitive and Advanced New LC-MS/MS Platform, Xevo TQ-S, in a Regulated BA Lab”

II. Paul Rainville

Senior Manager of Scientific Operations, ESD, Pharmaceutical & Life Sciences Business Operations. Waters Corporation

“Advances in Front-End Technologies for Bioanalysis”

Program

5:30 pm Social and registration
6:15 pm Complimentary dinner
7:00 pm Welcome and opening remarks
7:05 pm Xinping Fang
8:00 pm Paul Rainville
8:55 pm Closing remarks

Abstract for Talk II:

Modern bioanalytical chemists are charged with the task of developing high sensitivity assays for drug candidates with low circulating concentration, typically in the single pg/mL range. If this challenge was not difficult enough new economic, study design and ethical pressures demand that increasingly small volumes of samples are required and new sample formats such as dried blood spots. In this paper we will discuss the relative merits and pitfalls of two-dimensional liquid chromatography for bioanalytical applications when coupled to tandem quadrupole MS. Two-dimensional chromatography was evaluated for two applications i) to improve analyte cleanliness, thus increasing assay sensitivity and ii) to allow the direct injection of organic solvents onto a reversed phase system. As part of the study the mode of 2D operation was evaluated; forward flush, back flush and heart cut. Also we will discuss the use of a micro fabricated ceramic device packed with sub 2ìm for the analysis of plasma, bile and urine samples. We will show how the use of the micro scale separations device has been employed for the analysis of both small and large molecules.

Past MSDG Events