Mass spectrometric analysis of UV-crosslinked protein-nucleic acid complexes

The DNA-binding domains of E. coli uracil-DNA glycosylase (Ung) and human replication protein A (hRPA) were studied using a general protocol developed in our laboratory for probing protein-DNA interactions. The procedure involves purification and mass spectrometric analysis of the nucleopeptide-products of a tryptically digested UV-crosslinked protein-nucleic acid complex. In the case of Ung x dT₂₀ nucleoprotein complex, three nucleopeptide isomers having the same peptide backbone (T₁₈ peptide) but with dinucleotides attached to different aminoacids were separated. The tandem mass spectra from the isomers provided new structural information about Ung binding to DNA. Specifically, His₁₈₇, Ser₁₈₉, and His₁₉₄ from T₁₈ nucleopeptide were putatively identified as sites that photocrosslink to dT₂₀. Photochemical crosslinking of hRPA to oligonucleotide dT₃₀ produced two covalent hRPA70 x dT₃₀ complexes involving one of the protein's subunits (hRPA7O). Three crosslinked tryptic peptides were isolated using the same protocol as used for Ung and MALDI-TOF and nanoLC-ESI-MS/MS analyses revealed the identity of these peptides as T₄₃, T₂₈/₂₉, and a truncated *T₂₄/₂₅ (without the last 5 aminoacids from the C-teminal). Additional experiments showed that at least one amino acid from the sequence 383-VSDF-386 (located in T43), at least one residue from 235-ATAFNE-240 (*T₂₄/₂₅), and at least one residue from F269/T270 (T₂₈/₂₉) is involved in crosslinking. Aromatic residues contained in these peptides (F23 8, F269 and F386), which can form base stacking interactions with the DNA, are the residues most likely to be involved in crosslinking. These observations are in good agreement with previously published data regarding the single stranded-DNA binding site of hRPA obtained from crystal structure and from site-directed mutagenesis experiments

Liquid Chromatography Methods for Analysis of mRNA Poly(A) Tail Length and Heterogeneity

Messenger RNA (mRNA) is a new class of therapeutic compounds. The current advances in mRNA technology require the development of efficient analytical methods. In this work, we describe the development of several methods for measurement of mRNA poly(A) tail length and heterogeneity. Poly(A) tail was first cleaved from mRNA with the RNase T1 enzyme. The average length of a liberated poly(A) tail was analyzed with the size exclusion chromatography method. Size heterogeneity of the poly(A) tail was estimated with high-resolution ion-pair reversed phase liquid chromatography (IP RP LC). The IP RP LC method provides resolution of poly(A) tail oligonucleotide variants up to 150 nucleotide long. Both methods use a robust ultraviolet detection suitable for mRNA analysis in quality control laboratories. The results were confirmed by the LC-mass spectrometry (LC MS) analysis of the same mRNA sample. The poly(A) tail length and heterogeneity results were in good agreement

The 64-Kilodalton Capsid Protein Homolog of Beet Yellows Virus Is Required for Assembly of Virion Tails

The filamentous virion of the closterovirus Beet yellows virus (BYV) consists of a long body formed by the major capsid protein (CP) and a short tail composed of the minor capsid protein (CPm) and the virus-encoded Hsp70 homolog. By using nano-liquid chromatography-tandem mass spectrometry and biochemical analyses, we show here that the BYV 64-kDa protein (p64) is the fourth integral component of BYV virions. The N-terminal domain of p64 is exposed at the virion surface and is accessible to antibodies and mild trypsin digestion. In contrast, the C-terminal domain is embedded in the virion and is inaccessible to antibodies or trypsin. The C-terminal domain of p64 is shown to be homologous to CP and CPm. Mutation of the signature motifs of capsid proteins of filamentous RNA viruses in p64 results in the formation of tailless virions, which are unable to move from cell to cell. These results reveal the dual function of p64 in tail assembly and BYV motility and support the concept of the virion tail as a specialized device for BYV cell-to-cell movement

Enhanced Detection of Low-Abundance Host Cell Protein Impurities in High-Purity Monoclonal Antibodies Down to 1 ppm Using Ion Mobility Mass Spectrometry Coupled with Multidimensional Liquid Chromatography

The enormous dynamic range of proteinaceous species present in protein biotherapeutics poses a significant challenge for current mass spectrometry (MS)-based methods to detect low-abundance HCP impurities. Previously, an HCP assay based on two-dimensional chromatographic separation (high pH/low pH) coupled to high-resolution quadrupole time-of-flight (QTOF) mass spectrometry and developed in the author’s laboratory has been shown to achieve a detection limit of about 50 ppm (parts per milion) for the identification and quantification of HCPs present in monoclonal antibodies following Protein A purification. To improve the HCP detection limit we have explored the utility of several new analytical techniques for HCP analysis and thereby developed an improved liquid chromatography–mass spectrometry (LC–MS) methodology for enhanced detection of HCPs. The new method includes (1) the use of a new charge-surface-modified (CSH) C18 stationary phase to mitigate the challenges of column saturation, peak tailing, and distortion that are commonly observed in the HCP analysis; (2) the incorporation of traveling-wave ion mobility (TWIM) separation of coeluting peptide precursors, and (3) the improvement of fragmentation efficiency of low-abundance HCP peptides by correlating the collision energy used for precursor fragmentation with their mobility drift time. As a result of these improvements, the detection limit of the new methodology was greatly improved, and HCPs present at a concentration as low as 1 ppm (1 ng HCP/mg mAb) were successfully identified and quantified. The newly developed method was applied to analyze two high-purity mAbs (NIST mAb and Infliximab) expressed in a murine cell line. For both samples, low-abundance HCPs (down to 1 ppm) were confidently identified, and the identities of the HCPs were further confirmed by targeted MS/MS experiments. In addition, the performance of the assay was evaluated by an interlaboratory study in which three independent laboratories performed the same HCP assay on the mAb sample. The reproducibility of this assay is also discussed

Cross-Linking Mass Spectrometry and Mutagenesis Confirm the Functional Importance of Surface Interactions between CYP3A4 and Holo/Apo Cytochrome <i>b</i>₅

Cytochrome <i>b</i>₅ (cyt <i>b</i>₅) is one of the key components in the microsomal cytochrome P450 monooxygenase system. Consensus has not been reached about the underlying mechanism of cyt <i>b</i>₅ modulation of CYP catalysis. Both cyt <i>b</i>₅ and apo <i>b</i>₅ are reported to stimulate the activity of several P450 isoforms. In this study, the surface interactions of both holo and apo <i>b</i>₅ with CYP3A4 were investigated and compared for the first time. Chemical cross-linking coupled with mass spectrometric analysis was used to identify the potential electrostatic interactions between the protein surfaces. Subsequently, the models of interaction of holo/apo <i>b</i>₅ with CYP3A4 were built using the identified interacting sites as constraints. Both cyt <i>b</i>₅ and apo <i>b</i>₅ were predicted to bind to the same groove on CYP3A4 with close contacts to the B–B′ loop of CYP3A4, a substrate recognition site. Mutagenesis studies further confirmed that the interacting sites on CYP3A4 (Lys96, Lys127, and Lys421) are functionally important. Mutation of these residues reduced or abolished cyt <i>b</i>₅ binding affinity. The critical role of Arg446 on CYP3A4 in binding to cyt <i>b</i>₅ and/or cytochrome P450 reductase was also discovered. The results indicated that electrostatic interactions on the interface of the two proteins are functionally important. The results indicate that apo <i>b</i>_<i>5</i> can dock with CYP3A4 in a manner analogous to that of holo <i>b</i>₅, so electron transfer from cyt <i>b</i>₅ is not required for its effects