https://link.springer.com/content/pdf/10.1016/1044-0305(94)80016-2.pdf

An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.

Peptide sequence analysis using a combination of gas-phase ion/ion chemistry and tandem mass spectrometry (MS/MS) is demonstrated. Singly charged anthracene anions transfer an electron to multiply protonated peptides in a radio frequency quadrupole linear ion trap (QLT) and induce fragmentation of the peptide backbone along pathways that are analogous to those observed in electron capture dissociation. Modifications to the QLT that enable this ion/ion chemistry are presented, and automated acquisition of high-quality, single-scan electron transfer dissociation MS/MS spectra of phosphopeptides separated by nanoflow HPLC is described.

/pdf/peptide-and-protein-sequence-analysis-by-electron-transfer-2sqn15vkex.pdf

Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry

Methods in Lignin Chemistry

▪ Abstract A decade after the discovery of electrospray and matrix-assisted laser desorption ionization (MALDI), methods that finally allowed gentle ionization of large biomolecules, mass spectrometry has become a powerful tool in protein analysis and the key technology in the emerging field of proteomics. The success of mass spectrometry is driven both by innovative instrumentation designs, especially those operating on the time-of-flight or ion-trapping principles, and by large-scale biochemical strategies, which use mass spectrometry to detect the isolated proteins. Any human protein can now be identified directly from genome databases on the basis of minimal data derived by mass spectrometry. As has already happened in genomics, increased automation of sample handling, analysis, and the interpretation of results will generate an avalanche of qualitative and quantitative proteomic data. Protein-protein interactions can be analyzed directly by precipitation of a tagged bait followed by mass spectrometri...

Analysis of proteins and proteomes by mass spectrometry

According to Genome Sequencing Project statistics (http://www.ncbi.nlm.nih.gov/genomes/static/gpstat.html), as of Feb 16, 2012, complete gene sequences have become available for 2816 viruses, 1117 prokaryotes, and 36 eukaryotes.1–2 The availability of full genome sequences has greatly facilitated biological research in many fields, and has greatly contributed to the growth of proteomics.

Proteins are important because they are the direct bio-functional molecules in the living organisms. The term “proteomics” was coined from merging “protein” and “genomics” in the 1990s.3–4 As a post-genomic discipline, proteomics encompasses efforts to identify and quantify all the proteins of a proteome, including expression, cellular localization, interactions, post-translational modifications (PTMs), and turnover as a function of time, space and cell type, thus making the full investigation of a proteome more challenging than sequencing a genome. There are possibly 100,000 protein forms encoded by the approximate 20,235 genes of the human genome,5 and determining the explicit function of each form will be a challenge.

The progress of proteomics has been driven by the development of new technologies for peptide/protein separation, mass spectrometry analysis, isotope labeling for quantification, and bioinformatics data analysis. Mass spectrometry has emerged as a core tool for large-scale protein analysis. In the past decade, there has been a rapid advance in the resolution, mass accuracy, sensitivity and scan rate of mass spectrometers used to analyze proteins. In addition, hybrid mass analyzers have been introduced recently (e.g. Linear Ion Trap-Orbitrap series6–7) which have significantly improved proteomic analysis.

“Bottom-up” protein analysis refers to the characterization of proteins by analysis of peptides released from the protein through proteolysis. When bottom-up is performed on a mixture of proteins it is called shotgun proteomics,8–10 a name coined by the Yates lab because of its analogy to shotgun genomic sequencing.11 Shotgun proteomics provides an indirect measurement of proteins through peptides derived from proteolytic digestion of intact proteins. In a typical shotgun proteomics experiment, the peptide mixture is fractionated and subjected to LC-MS/MS analysis. Peptide identification is achieved by comparing the tandem mass spectra derived from peptide fragmentation with theoretical tandem mass spectra generated from in silico digestion of a protein database. Protein inference is accomplished by assigning peptide sequences to proteins. Because peptides can be either uniquely assigned to a single protein or shared by more than one protein, the identified proteins may be further scored and grouped based on their peptides. In contrast, another strategy, termed ‘top-down’ proteomics, is used to characterize intact proteins (Figure 1). The top-down approach has some potential advantages for PTM and protein isoform determination and has achieved notable success. Intact proteins have been measured up to 200 kDa,12 and a large scale study has identified more than 1,000 proteins by multi-dimensional separations from complex samples.13 However, the top-down method has significant limitations compared with shotgun proteomics due to difficulties with protein fractionation, protein ionization and fragmentation in the gas phase. By relying on the analysis of peptides, which are more easily fractionated, ionized and fragmented, shotgun proteomics can be more universally adopted for protein analysis. In fact, a hybrid of bottom-up and top-down methodologies and instrumentation has been introduced as middle-down proteomics.14 Essentially, middle-down proteomics analyzes larger peptide fragments than bottom-up proteomics, minimizing peptide redundancy between proteins. Additionally the large peptide fragments yield similar advantages as top-down proteomics, such as gaining further insight into post-translational modifications, without the analytical challenges of analyzing intact proteins. Shotgun proteomics has become a workhorse for the analysis of proteins and their modifications and will be increasingly combined with top-down methods in the future.



Figure 1

Proteomic strategies: bottom-up vs. top-down vs. middle-down. The bottom-up approach analyzes proteolytic peptides. The top-down method measures the intact proteins. The middle-down strategy analyzes larger peptides resulted from limited digestion or ...



In the past decade shotgun proteomics has been widely used by biologists for many different research experiments, advancing biological discoveries. Some applications include, but are not limited to, proteome profiling, protein quantification, protein modification, and protein-protein interaction. There have been several reviews nicely summarizing mass spectrometry history,15 protein quantification with mass spectrometry,16 its biological applications,5,17–26 and many recent advances in methodology.27–32 In this review, we try to provide a full and updated survey of shotgun proteomics, including the fundamental techniques and applications that laid the foundation along with those developed and greatly improved in the past several years.

Protein Analysis by Shotgun/Bottom-up Proteomics

Recent improvements in and analytical applications of advanced ion trap technology

Instrumentation, applications, and energy deposition in quadrupole ion-trap tandem mass spectrometry

Pulsed positive negative ion chemical ionization mass spectrometry

Operation of a quadrupole ion trap mass spectrometer to achieve high mass/charge ratios

In a quadrupole ion store or ion trap type mass spectometer, significantly improved mass selection is achieved by simultaneously trapping ions within the mass range of interest and then scanning the applied RF and DC voltages or the frequency ω to sequentially render unstable trapped ions of consecutive specific masses. These are passed out through apertures in an end cap to a high gain electron multiplier to provide a signal indicative of the ion mass. Sensitivity and mass resolution is also enhanced by operating the ion trap at a relatively high pressure in the range 1×10 -1 to 1×10 -5 torr. The presence of collision gas molecules, such as helium, improves sensitivity and mass resolution. In addition, the structure itself is built of stacked units, sealed by O-rings, which are easily disassembled for cleaning.

George C. Stafford

Papers

Recent improvements in and analytical applications of advanced ion trap technology

Instrumentation, applications, and energy deposition in quadrupole ion-trap tandem mass spectrometry

Pulsed positive negative ion chemical ionization mass spectrometry

Operation of a quadrupole ion trap mass spectrometer to achieve high mass/charge ratios

Method of mass analyzing a sample by use of a quadrupole ion trap