Using high energy photons, such as in the UV range, results in high energy deposition per photon and thus translates into a greater array of fragment ions. 1618For example, photoexcitation using 157 nm or 193 nm photons accesses excited electronic states, thus opening new fragmentation pathways of peptides or proteins. The number of photons needed to induce dissociation is dependent on the photon wavelength and can range from several hundred photons in the IR range (~0. 1 eV per photon) to a single UV photon (~38 eV per photon). played such a critical GDC-0032 (Taselisib) role in the fields of biochemistry, molecular biology, medicinal chemistry, biotechnology, and structural biology. Moreover, unravelling the fundamental underpinnings of these activation methods have shed light on the factors that influence ion fragmentation upon energization, thus providing predictive insight and motivating new strategies that capitalize on manipulating ion dissociation behavior intended for specific applications. Given the critical role that tandem mass spectrometry has played in the field of proteomics and structural biology, this review will emphasize the ion activation methods that have been used to analyze peptides and proteins with an emphasis on new applications over the past three years. There are numerous excellent review and tutorial articles that have focused on mass spectrometry-based proteomics technologies, proteomic applications, and specific activation methods in recent years, and thus readers are directed to these to provide additional perspectives. 124In addition, a recent review focused specifically on activation methods in proteomics with an emphasis on characterization of post-translational modifications and tandem mass spectrometry methods for quantitation, 7so these topics are not covered here. This review opens with some basic tutorial sections to provide background information, followed by more specialized sub-topics that demonstrate some of the more recent high impact applications of activation methods for peptides and proteins. == Tandem Mass Spectrometry and Proteomics == Tandem mass spectrometry, known as MS/MS, is one of most versatile and powerful methods for acquiring structural information about a molecule. Although the elemental composition of a peptide can be determined based on highly accurate mass measurement, high mass accuracy only is not sufficient to assign a sequence or to differentiate peptide isomers which have the same elemental and amino acid compositions. This means that sequence-specific information afforded by MS/MS is GDC-0032 (Taselisib) indispensable for peptide and protein analysis. The general process of tandem mass spectrometry involves isolation/selection and manipulation GDC-0032 (Taselisib) (via energization or reaction) of a population of precursor ions and detection of the resulting products. Ion activation, not reaction, is the focus of this review, and in this respect energy can be added in multiple small actions primarily in vibrational modes, as is the case for low energy collisional activation, or in a single fast event, such as absorption of a UV photon. As such, the rate and amount of energy deposition, as well as mechanistic effects, can have significant impact on the outcome for peptides and proteins in terms of the types, abundances, and range of fragment ions produced. Intended for even more structural information, fragment ions can be subsequently activated and dissociated multiples times in a process called MSn. There are several general categories of activation methods that have been used for analysis IGFBP1 of peptides and proteins, including ones based on collisions with gas molecules or surfaces, interactions with electrons or electron-donating or electron-accepting reagent ions, or absorption of photons, all of which will be covered in this review, in addition to several emerging methods that may gain popularity as they are more thoroughly developed. Many of these activation methods are complementary to one another, thus providing supplemental information when used in a cooperative manner. In fact , no single activation method has proven to be universal for all mass spectrometry platforms and all types of molecules which is why new activation methods continue to be explored and existing methods continue to be refined. Tandem mass spectrometry has been widely applied for characterization of individual peptides or proteins, in addition to broader, deeper, higher throughput studies for proteomics. 124In the context of mass spectrometry-based proteomics, there are three main approaches: GDC-0032 (Taselisib) bottom-up, middle-down, and top-down (Figure 1). In the very popular bottom-up approach, proteins are enzymatically digested to produce more readily analyzed peptides that are representative of the original proteins. 14The peptides are typically separated using high performance liquid chromatography (or other emerging separation methods like capillary electrophoresis), ionized, and activated to create informative fragmentation patterns. Various algorithms are used to assign fragment ions of each peptide which are then matched to proteins. Alternatively, using a top-down approach, proteins are not enzymatically digested but rather are.