scholarly journals Recent Progress in FD-LC-MS/MS Proteomics Method

2021 ◽  
Vol 9 ◽  
Author(s):  
Hiroshi Kobayashi ◽  
Kazuhiro Imai
Keyword(s):  
Mass Spectrometry ◽  
Protein Identification ◽  
Intact Cell ◽  
Intact Protein ◽  
Identification Algorithm ◽  
Differential Proteomics ◽  
Proteomics Analysis ◽  
Peak Separation ◽  
Two Samples ◽  
Fluorogenic Derivatization

Through the course of our bio-analytical chemistry studies, we developed a novel proteomics analysis method, FD-LC-MS/MS (fluorogenic derivatization-liquid chromatography-tandem mass spectrometry). This method consists of fluorogenic derivatization (FD), LC separation, and detection/quantification of the derivatized proteins, followed by isolation, tryptic digestion of the isolated proteins, and final identification of the isolated proteins using electrospray ionization nano-LC-MS/MS of the generated peptide mixtures with a probability-based protein identification algorithm. In this review, we will present various examples where this method has been used successfully to identify expressed proteins in individual human cells. FD-LC-MS/MS is also suitable for differential proteomics analysis. Here, two biological samples are treated by the same steps mentioned above, and the two chromatograms obtained are compared to identify peaks with different intensities (variation in protein levels). Associated peak fractions are then isolated, and the differentially expressed proteins between the two samples are identified by LC-MS/MS. Several biomarkers for cancers have been identified by FD-LC-MS/MS. For more efficient separation, nano-flow LC with a phenyl-bonded monolithic silica-based capillary column was adopted for cell-expressed intact protein analysis. The derivatized human cell proteins (K562) and yeast cell (Saccharomyces cerevisiae) proteins as model intact cell proteins were analyzed by nano-flow LC with fluorescence detection. More than 1,300 protein peaks were separated/detected from both cells. For straightforward comparison of multiple peak separation profiles, a novel type of chromatogram display, termed the “spiderweb” chromatogram, was developed. A nano-LC-FD-LC-mass spectrometry trial for molecular weight estimation of FD proteins has also been conducted.

scholarly journals On the utility of predictive chromatography to complement mass spectrometry based intact protein identification

2011 ◽  
Vol 402 (8) ◽  
pp. 2521-2529 ◽  
Author(s):  
Marina L. Pridatchenko ◽  
Tatyana Yu. Perlova ◽  
Hisham Ben Hamidane ◽  
Anton A. Goloborodko ◽  
Irina A. Tarasova ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Identification ◽  
Intact Protein

scholarly journals SQID: An Intensity-Incorporated Protein Identification Algorithm for Tandem Mass Spectrometry

2011 ◽  
Vol 10 (4) ◽  
pp. 1593-1602 ◽  
Author(s):  
Wenzhou Li ◽  
Li Ji ◽  
Jonathan Goya ◽  
Guanhong Tan ◽  
Vicki H. Wysocki
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Protein Identification ◽  
Identification Algorithm ◽  
Tandem Mass

scholarly journals In situ mass spectrometry analysis of intact proteins and protein complexes from biological substrates

2020 ◽  
Vol 48 (1) ◽  
pp. 317-326
Author(s):  
Oliver J. Hale ◽  
Helen J. Cooper
Keyword(s):  
Mass Spectrometry ◽  
Protein Identification ◽  
Rapid Development ◽  
Ion Sources ◽  
Mass Spectrometry Analysis ◽  
Intact Protein ◽  
Intact Proteins ◽  
Electrospray Ionisation ◽  
Biological Substrates

Advances in sample preparation, ion sources and mass spectrometer technology have enabled the detection and characterisation of intact proteins. The challenges associated include an appropriately soft ionisation event, efficient transmission and detection of the often delicate macromolecules. Ambient ion sources, in particular, offer a wealth of strategies for analysis of proteins from solution environments, and directly from biological substrates. The last two decades have seen rapid development in this area. Innovations include liquid extraction surface analysis, desorption electrospray ionisation and nanospray desorption electrospray ionisation. Similarly, developments in native mass spectrometry allow protein–protein and protein–ligand complexes to be ionised and analysed. Identification and characterisation of these large ions involves a suite of hyphenated mass spectrometry techniques, often including the coupling of ion mobility spectrometry and fragmentation techniques. The latter include collision, electron and photon-induced methods, each with their own characteristics and benefits for intact protein identification. In this review, recent developments for in situ protein analysis are explored, with a focus on ion sources and tandem mass spectrometry techniques used for identification.

Mass Spectrometry Techniques: Principles and Practices for Quantitative Proteomics

2020 ◽  
Vol 21 ◽  
Author(s):  
Rocco J. Rotello ◽  
Timothy D. Veenstra
Keyword(s):  
Mass Spectrometry ◽  
Quantitative Proteomics ◽  
Protein Identification ◽  
Dynamic Nature ◽  
Complete Coverage ◽  
The Past ◽  
Proteome Level ◽  
A Cell ◽  
Quantitative Changes ◽  
Made In

: In the current omics-age of research, major developments have been made in technologies that attempt to survey the entire repertoire of genes, transcripts, proteins, and metabolites present within a cell. While genomics has led to a dramatic increase in our understanding of such things as disease morphology and how organisms respond to medications, it is critical to obtain information at the proteome level since proteins carry out most of the functions within the cell. The primary tool for obtaining proteome-wide information on proteins within the cell is mass spectrometry (MS). While it has historically been associated with the protein identification, developments over the past couple of decades have made MS a robust technology for protein quantitation as well. Identifying quantitative changes in proteomes is complicated by its dynamic nature and the inability of any technique to guarantee complete coverage of every protein within a proteome sample. Fortunately, the combined development of sample preparation and MS methods have made it capable to quantitatively compare many thousands of proteins obtained from cells and organisms.

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