Most recent mass spectrometry-based workflows have focused on analysis of synthetic oligonucleotide therapeutics. To date, several nucleic acid-based drugs have been approved for use by the FDA, with many more currently undergoing phase-II and -III clinical trials, with the potential to treat a broad range of diseases and previously undruggable targets. Among those platforms approved for therapeutic use are antisense oligonucleotides, siRNAs, miRNAs, and transcriptional gene activation -all alongside the notable mRNA vaccines deployed to combat the COVID-19 pandemic in 2021 . In addition to these modalities, approaches such as RNA-guided CRISPR/Cas9 gene editing are likewise being developed and show promise in their therapeutic effects. These molecules can range from short (< 30 nt) oligonucleotides in the case of antisense strands to very long (> 1000 nt) mRNA strands. Regardless of their size or function, each therapeutic must be well-characterized to ensure their efficacy and stability, including characterization of the suite of chemical modifications employed to enhance their functions. Synthetic oligonucleotides often include non-canonical modifications, including phosphorothioate backbones 3 and 2'O to 4' bridged sugars 4 , both imparting resistance to enzymatic degradation, and other sugar modifications like 2' ethyl-O-methylation. The landscape of post-transcriptional modifications present on biological nucleic acids is far more extensive, with some 170 identified to date in RNA alone, each of which can co-occur on a given strand. Common modifications include methylation of the nucleotide sugars or nitrogenous bases, along with linkage isomers like pseudouridine, and some non-canonical bases like wybutosine or inosine. tRNAs are among some of the earliest nucleic acids studied by mass spectrometry with a focus on deciphering posttranscriptional modifications. These oligonucleotides often contain multiple modifications in a single sequence, each of which can be critical to its resulting function, and these are modulated based on the cell's response to extracellular stimuli. To characterize post-transcriptional modifications via mass spectrometry, large nucleic acids (> 50 nt) are most often digested using an endonuclease and the resulting oligonucleotides separated using liquid chromatography prior to MS or MS/MS analysis. This approach is analogous to "bottom-up" approaches in proteomics, though the methodologies employed for nucleic acids tend to be less developed. These bottom-up methods can be highly effective but are often unable to decipher different permutations of covalent modification which can be biologically relevant. As such, top-down methods, where the intact biopolymer is ionized, fragmented, and analyzed by MS without prior digestion, have spurred interest and led to successful outcomes in proteomics and are being extended to nucleic acids. Comprehensive analysis of smaller nucleic acids (< 100 nt) is within reach, and larger mRNAs and lncRNAs (upwards of 1000 nt) remain an intriguing future goal. tRNAs were some of the first relatively large oligonucleotides characterized by mass spectrometry, and recent interest has largely focused on nucleic acid therapeutics -including small (< 25 nt) DNAs and RNAs, 10 structured oligonucleotides like G-quadruplexes, as well as larger guide RNA molecules used in CRISPR/Cas9 gene editing platforms. Recent studies reporting MS for guide RNA characterization have reported improvements in topdown workflows, optimizing the conditions for ionization, fragmentation, and data processing. One notable challenge was effective desalting of progressively larger nucleic acids. The characteristic phosphodiester backbone is very acidic, making it amenable to negative mode ionization. This acidic backbone also exhibits high affinity for binding small cations, such as sodium and ammonium, present in the electrospray solution and can be retained during the ionization process. Often, the resulting adducts increase spectral complexity and disperse analyte signal across multiple m/z values, with longer nucleic acids exhibiting progressively more adducts. To mitigate cationic adducts observed in mass spectra of nucleic acids, two main approaches have been developed to effectively "desalt" the sample prior to its introduction to the mass spectrometer. Both methods employ liquid chromatography but differ in mobile phase composition; either ion pairing reagents or basic additives enable efficient on-line desalting of oligonucleotides. Recently, piperidine has been used to achieve highly effective desalting via competitive hydrogen bonding with the phosphodiester backbone of large (100 nt) gRNA molecules. To characterize these large nucleic acids, MS/MS is used to induce backbone cleavage to produce sequence-informative fragment ions. Collision-induced dissociation (CID) causes backbone cleavages for even very large nucleic acids, yielding high sequence coverage. Photon-based dissociation methods have also been employed to characterize small oligonucleotides. The performance of ultraviolet photodissociation (UVPD) using 193 nm or 213 nm ultraviolet photons have proven effective for these small nucleic acids, and 213 nm UVPD was also effective for characterizing larger gRNA molecules. Activated-ion electron photodetachment dissociation (a-EPD), a method in which charge-reduced precursor ions generated during photoactivation are re-isolated and subsequently activated by CID, has also been successful for characterizing a range of RNA and DNA molecules. Another critical aspect of the workflow is the interpretation of the fragment ions to construct the sequence and identify/localize modifications. While new informatics approaches have begun to address the challenges inherent in nucleic acid characterization, these lag behind their counterparts in proteomics both in sophistication and accessibility. Here we report an integrated workflow incorporating a nanoscale online desalting platform for analysis of an array of nucleic acids, MS/MS analysis of modified nucleic acids, as illustrated for tRNA isoforms, and automated fragment identification directly in the m/z domain.