Integrated Microfluidic System Enabling Protein Digestion, Peptide Separation, and Protein Identification

2001 ◽  
Vol 73 (11) ◽  
pp. 2648-2655 ◽  
Author(s):  
Jun Gao ◽  
Jingdong Xu ◽  
Laurie E. Locascio ◽  
Cheng S. Lee
Keyword(s):  
Protein Identification ◽  
Microfluidic System ◽  
Protein Digestion ◽  
Peptide Separation

scholarly journals Vibratory Reaction Unit for the Rapid Analysis of Proteins and Glycochains

2007 ◽  
Vol 2 ◽  
pp. 117739010700200 ◽  
Author(s):  
Yukie Sasakura ◽  
Makoto Nogami ◽  
Noriko Kobayashi ◽  
Katsuhiro Kanda
Keyword(s):  
Mass Spectrometry ◽  
Solid Surface ◽  
Protein Identification ◽  
Immobilized Enzymes ◽  
Rapid Analysis ◽  
Single Step ◽  
Protein Digestion ◽  
Sequence Coverage ◽  
Micro Scale ◽  
Reaction Unit

A protein digestion system using immobilized enzymes for protein identification and glycochain analyses has been developed, and a vibration reaction unit for micro-scale sample convection on an enzyme-immobilized solid surface was constructed. BSA as a model substrate was digested by this unit, and was successfully identified by mass spectrometry (MS) analyses. Compared to the conventional liquid-phase digestion, the reaction unit increased the number of matched peptides from 9 to 26, protein score from 455 to 1247, and sequence coverage from 21% to 48%. Glycopeptidase F (NGF), an enzyme that cleaves N-glycans from glycoproteins, was also immobilized and used to remove the glycochains from human immunoglobulin G (IgG). Trypsin and NGF were immobilized on the same solid surface and used to remove glycochains from IgG in single-step. Glycochains were labeled with fluorescent reagent and analyzed by HPLC. Several peaks corresponding to the glycochains of IgG were detected. These results suggested that the single-step digestion system, by immobilized multiple enzymes (trypsin and NGF) would be effective for the rapid structural analysis of glycoproteins.

Multiwell in-gel protein digestion and microscale sample preparation for protein identification by mass spectrometry

PROTEOMICS ◽  
2002 ◽  
Vol 2 (2) ◽  
pp. 145-150 ◽  
Author(s):  
Malcolm G. Pluskal ◽  
Alla Bogdanova ◽  
Mary Lopez ◽  
Sara Gutierrez ◽  
Aldo M. Pitt
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Protein Identification ◽  
Protein Digestion

scholarly journals Influence of NanoLC Column and Gradient Length as well as MS/MS Frequency and Sample Complexity on Shotgun Protein Identification of Marine Bacteria

2017 ◽  
Vol 27 (3) ◽  
pp. 199-212 ◽  
Author(s):  
Lars Wöhlbrand ◽  
Ralf Rabus ◽  
Bernd Blasius ◽  
Christoph Feenders
Keyword(s):  
Marine Bacteria ◽  
Protein Identification ◽  
Shotgun Proteomics ◽  
Sample Complexity ◽  
Column Length ◽  
Peptide Separation ◽  
Total Analysis Time ◽  
Total Analysis ◽  
Nano Liquid Chromatography ◽  
Esi Mass Spectrometry

Protein identification by shotgun proteomics, i.e., nano-liquid chromatography (nanoLC) peptide separation online coupled to electrospray ionization (ESI) mass spectrometry (MS)/MS, is the most widely used gel-free approach in proteome research. While the mass spectrometer accounts for mass accuracy and MS/MS frequency, the nanoLC setup and gradient time influence the number of peptides available for MS analysis, which ultimately determine the number of proteins identifiable. Here, we report on the influence of (i) analytical column length (15, 25, or 50 cm) coupled to (ii) the applied gradient length (120, 240, 360, 480, or 600 min), as well as (iii) MS/MS frequency on peptide/protein identification by shotgun proteomics of (iv) 2 marine bacteria. Longer gradients increased the number of peptides/proteins identified as well as the reproducibility of identification. Furthermore, longer analytical columns strictly enlarge the covered proteome complement. Notably, the proteome complement identified with a short column and applying a long gradient is also covered when using longer columns with shorter gradients. Coverage of the proteome complement further increases with higher MS/MS frequency. Compilation of peptide lists of replicate analyses (same gradient length) improves protein identification, while compilation of analyses with different gradient lengths yields a similar or even higher number of proteins using comparable or even less total analysis time.

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