Intact Protein Analysis by Mass Spectrometry to Characterize the Truncated Hemoglobin THB1 from Chlamydomonas Reinhardtii

Mass spectrometry (MS) is the primary analytical tool used to characterize proteins within the biopharmaceutical industry. Electrospray ionization (ESI) coupled to liquid chromatography is the gold standard technique for intact protein analysis. However, speed limitations prevent analysis of large sample numbers (>1000) in a day. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) MS, an ambient ionization MS technology, has recently been established as a platform for high throughput small molecule analysis. Here we report the application of such system for intact protein analysis. Up to 150 kDa proteins are detectable on a high throughput IR-MALDESI-MS system and we have evaluated how matrix affects signal. The system can analyze up to 22 protein samples in a second. Protein autophosphorylation, compound binding kinetics and compound modifications to a probe protein are demonstrated as applications. Top-down protein sequencing was conducted to identify a cysteine modification site. Two plate-based high throughput sample cleanup methods were coupled to IR-MALDESI-MS to enable analysis of samples containing high amounts of salts and buffers without compromising speed.

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Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis – electrospray – time-of-flight mass spectrometry

Electrophoresis ◽

10.1002/elps.200600075 ◽

2006 ◽

Vol 27 (13) ◽

pp. 2638-2650 ◽

Cited By ~ 100

Author(s):

Elvira Balaguer ◽

Uwe Demelbauer ◽

Matthias Pelzing ◽

Victoria Sanz-Nebot ◽

José Barbosa ◽

...

Keyword(s):

Mass Spectrometry ◽

Capillary Electrophoresis ◽

Time Of Flight ◽

Protein Analysis ◽

Intact Protein ◽

Flight Mass Spectrometry ◽

Intact Protein Analysis

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Synthesis and Matrix Properties of α-Cyano-5-phenyl-2,4-pentadienic Acid (CPPA) for Intact Proteins Analysis by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Molecules ◽

10.3390/molecules25246054 ◽

2020 ◽

Vol 25 (24) ◽

pp. 6054

Author(s):

Antonio Monopoli ◽

Angelo Nacci ◽

Tommaso R. I. Cataldi ◽

Cosima D. Calvano

Keyword(s):

Mass Spectrometry ◽

Laser Desorption ◽

Protein Analysis ◽

Ionization Mass Spectrometry ◽

Intact Protein ◽

Ionization Mass ◽

Laser Desorption Ionization ◽

Desorption Ionization ◽

Matrix Assisted Laser ◽

Matrix Assisted Laser Desorption

The effectiveness of a synthesized matrix, α-cyano-5-phenyl-2,4-pentadienic acid (CPPA), for protein analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in complex samples such as foodstuff and bacterial extracts, is demonstrated. Ultraviolet (UV) absorption along with laser desorption/ionization mass spectrometry (LDI-MS) experiments were systematically conducted in positive ion mode under standard Nd:YLF laser excitation with the aim of characterizing the matrix in terms of wavelength absorption and proton affinity. Besides, the results for standard proteins revealed that CPPA significantly enhanced the protein signals, reduced the spot-to-spot variability and increased the spot homogeneity. The CPPA matrix was successful employed to investigate intact microorganisms, milk and seed extracts for protein profiling. Compared to conventional matrices such as sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA) and 4-chloro-α-cyanocinnamic acid (CClCA), CPPA exhibited better signal-to-noise (S/N) ratios and a uniform response for most examined proteins occurring in milk, hazelnut and in intact bacterial cells of E. coli. These findings not only provide a reactive proton transfer MALDI matrix with excellent reproducibility and sensitivity, but also contribute to extending the battery of useful matrices for intact protein analysis.

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