Formaldehyde has been used for decades to probe macromolecular structure and function and to trap complexes, cells, and tissues for further analysis. Formaldehyde crosslinking is routinely employed for detection and quantification of protein-DNA interactions, interactions between chromatin proteins, and interactions between distal segments of the chromatin fiber. Despite widespread use and a rich biochemical literature, important aspects of formaldehyde behavior in cells have not been well described. Here, we highlight features of formaldehyde chemistry relevant to its use in analyses of chromatin complexes, focusing on how its properties may influence studies of chromatin structure and function.Prior to its use in the chromatin field, formaldehyde use had a long history in a number of fields, including vaccine production (1, 2) and histology (3). In this review, we focus on its use in chromatin immunoprecipitation approaches and protein-protein interaction studies applied to understand the location and abundance of transcription factor binding along DNA. A recent complementary perspective highlights gaps in knowledge with a particular focus on how formaldehyde crosslinking data have been used to interpret aspects of chromatin three-dimensional organization (4). Here, we briefly review prior work describing formaldehyde reactivity toward proteins, DNA, and their constituent monomers. This information provides a basis for understanding how formaldehyde functions in widely used assays in the chromatin field, and conversely, highlights less well understood aspects of formaldehyde behavior in cells. These issues are of significance for designing crosslinkingbased studies as well as for properly interpreting the resulting data. The analysis of formaldehyde-fixed chromatin has provided fundamental insights into where and when regulatory factors associate with the DNA template in vivo, but it in general does not provide unambiguous information about chromatin binding kinetics. A major goal of ongoing work is to understand kinetic and thermodynamic aspects of chromatin complex assembly at single copy loci in vivo. Development of experimental strategies to achieve these goals will require a deeper and more comprehensive understanding of the effects mediated by formaldehyde in cells.The following discussion provides a framework for understanding aspects of formaldehyde function when used to trap macromolecular complexes in cells, with the main features shown in Fig. 1. Beginning with basic chemical reactivity, this review will explore the ability of formaldehyde to crosslink with proteins and DNA to form protein-protein or protein-DNA complexes, common molecular quenchers, and the potential for crosslink reversal. Progress in capturing crosslinked complexes will also be discussed with an emphasis on the impact of a better understanding of formaldehyde chemistry in vivo. Basic ChemistryFormaldehyde is the smallest aldehyde, an electrophilic molecule susceptible to chemical attack by a wide range of nucleophilic species of ...

Correct identification of protein post-translational modifications (PTMs) is crucial to understanding many aspects of protein function in biological processes. G-PTM-D is a recently developed technique for global identification and localization of PTMs. Spectral file calibration prior to applying G-PTM-D, and algorithmic enhancements in the peptide database search significantly increase the accuracy, speed, and scope of PTM identification. We enhance G-PTM-D by using multinotch searches and demonstrate its effectiveness in identification of numerous types of PTMs including high-mass modifications such as glycosylations. The changes described in this work lead to a 20% increase in the number of identified modifications and an order of magnitude decrease in search time. The complete workflow is implemented in MetaMorpheus, a software tool that integrates the database search procedure, identification of coisolated peptides, spectral calibration, and the enhanced G-PTM-D workflow. Multinotch searches are also shown to be useful in contexts other than G-PTM-D by producing superior results when used instead of standard narrow-window and open database searches.

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Human proteomic databases required for MS peptide identification are frequently updated and carefully curated, yet are still incomplete because it has been challenging to acquire every protein sequence from the diverse assemblage of proteoforms expressed in every tissue and cell type. In particular, alternative splicing has been shown to be a major source of this cell-specific proteomic variation. Many new alternative splice forms have been detected at the transcript level using next generation sequencing methods, especially RNA-Seq, but it is not known how many of these transcripts are being translated. Leveraging the unprecedented capabilities of next generation sequencing methods, we collected RNA-Seq and proteomics data from the same cell population (Jurkat cells) and created a bioinformatics pipeline that builds customized databases for the discovery of novel splicejunction peptides. Eighty million paired-end Illumina reads and ϳ500,000 tandem mass spectra were used to identify 12,873 transcripts (19,320 including isoforms) and 6810 proteins. We developed a bioinformatics workflow to retrieve high-confidence, novel splice junction sequences from the RNA data, translate these sequences into the analogous polypeptide sequence, and create a customized splice junction database for MS searching. Based on the RefSeq gene models, we detected 136,123 annotated and 144,818 unannotated transcript junctions. Of those, 24,834 unannotated junctions passed various quality filters (e.g. minimum read depth) and these entries were translated into 33,589 polypeptide sequences and used for database searching. We discovered 57 splice junction peptides not present in the Uniprot-Trembl proteomic database comprising an array of different splicing events, including skipped exons, alternative donors and acceptors, and noncanonical transcriptional start sites. To our knowledge this is the first example of using sample-specific RNA-Seq data to create a splice-junction database and discover new peptides resulting from alternative splicing. Mass spectrometry-based proteomics relies on accurate databases to identify and quantify proteins, including those derived from splice variants, indels, and single nucleotide variants (SNVs) 1 (1). Most computational search algorithms detect peptides by scoring the degree of similarity between in silico derived and experimental peptide spectra, and thus can only identify peptides that are present in the proteomic database. If the polypeptide sequence is not present in the database used for searching, even if the peptide is present in the sample, it will fail to be detected.Human proteomic databases used for mass spectrometric peptide identification are frequently updated and carefully curated, yet are still incomplete. Despite efforts to comprehensively annotate every gene product, there are still many undiscovered proteoforms (2) because the complete human proteome-the aggregate of all protein products expressed in every tissue, cell, and cellular state-turns out to be vastly more complex than was...

Monolayers of the polypeptide pofy(L-lysme) (PL) are used to control the specific adsorption of proteins onto gold surfaces. A PL monolayer modified with biotin is electrostatically adsorbed onto a vapor-deposited gold film that has been coated with a self-assembled monolayer of the alkanethiol 11-mercaptoundecanoic acid (MUA). The immobilized biotin moieties act as specific adsorption sites for the protein avidin. Adsorption of the biopolymers onto the gold surface is monitored with a combination of surface plasmon resonance (SPR) and fluorescence measurements. By varying the percent biotinylation of the lysine residues on the PL prior to deposition, the surface coverage of avidin can be controlled to create either full or partial monolayers. The thickness of a full monolayer of avidin is 41 A, as determined by the SPR measurements. At high surface coverages of avidin, an excess of biotin sites is required to overcome steric hindrance. The PL monolayer and any adsorbed avidin can be easily rinsed from the surface with a low or high pH solution. This removal allows for quantitation of the adsorbed molecules by fluorescence measurements in solution rather than on the gold surface. In this manner, fluorescein-labeled PL and avidin are used to determine absolute surface coverages of 4 x 1014 lysine residues cm-2 for the PL monolayer and 3 x 1012 avidin molecules cm-2 for the full avidin monolayer. SPR imaging experiments are employed to verify that UV photopatteming of the MUA/PL bilayers can be used to spatially direct the adsorption of avidin onto the gold surface. The polyfysine attachment methodology will be beneficial in the fabrication of adsorption biosensors.The fabrication of bioanalytical devices such as adsorption biosensors, enzyme-coated electrodes, and affinity chromatography columns often requires the attachment of protein molecules onto a solid surface.1-3 In this attachment step, it is frequently necessary to control the specific adsorption of a particular protein and to prevent the nonspecific adsorption of other biological