Limits...
Native Liquid Extraction Surface Analysis Mass Spectrometry: Analysis of Noncovalent Protein Complexes Directly from Dried Substrates.

Martin NJ, Griffiths RL, Edwards RL, Cooper HJ - J. Am. Soc. Mass Spectrom. (2015)

Bottom Line: Here, we demonstrate native LESA mass spectrometry of noncovalent protein complexes of myoglobin and hemoglobin from a range of surfaces.Heme-bound dimers and monomers were also observed.The 'contact' LESA approach was particularly suitable for the analysis of hemoglobin tetramers from DBS.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

ABSTRACT
Liquid extraction surface analysis (LESA) mass spectrometry is a promising tool for the analysis of intact proteins from biological substrates. Here, we demonstrate native LESA mass spectrometry of noncovalent protein complexes of myoglobin and hemoglobin from a range of surfaces. Holomyoglobin, in which apomyoglobin is noncovalently bound to the prosthetic heme group, was observed following LESA mass spectrometry of myoglobin dried onto glass and polyvinylidene fluoride surfaces. Tetrameric hemoglobin [(αβ)2(4H)] was observed following LESA mass spectrometry of hemoglobin dried onto glass and polyvinylidene fluoride (PVDF) surfaces, and from dried blood spots (DBS) on filter paper. Heme-bound dimers and monomers were also observed. The 'contact' LESA approach was particularly suitable for the analysis of hemoglobin tetramers from DBS.

No MeSH data available.


Related in: MedlinePlus

Native LESA mass spectra of hemoglobin from dried blood spots: (a) Orbitrap mass analyzer; (b) Q-TOF mass analyzer; (c) ‘contact’ LESA with Q-TOF mass analyzer
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4494149&req=5

Fig4: Native LESA mass spectra of hemoglobin from dried blood spots: (a) Orbitrap mass analyzer; (b) Q-TOF mass analyzer; (c) ‘contact’ LESA with Q-TOF mass analyzer

Mentions: Figure 4 shows the results obtained following native LESA MS of dried blood spots on filter card. The concentration of hemoglobin in human blood is approximately 130 mg/mL for a healthy adult male and 120 mg/mL for a healthy adult female [38]. Results obtained from the Orbitrap (Figure 4a) were similar to those of the hemoglobin standard, showing the presence of (αβ)2H dimers, (αβ)H dimers, αH, βH, and unbound monomers. The relative abundance of the (αβ)2H dimer was greater from the DBS sample than the protein standard. This observation may be the result of reducing the inlet capillary temperature to 200°C. Tetramers were not observed in the Orbitrap data; however, these were observed in the Q-TOF mass spectrum (Figure 4b). Native LESA MS using the Q-TOF resulted in detection of (αβ)24H tetramers, αβ2H dimers, αβH dimers, αH, and βH. As for the standard protein, the tetramer peaks are broad (~100 Th) wide, but the relative abundance of the tetramers is much higher. Figure 4c shows the mass spectrum obtained following ‘contact’ LESA sampling [23] of DBS. The results suggest that this approach is particularly suitable for the analysis of Hb tetramers from DBS. The relative abundance of the peaks corresponding to the tetramers is greater than following standard LESA. In addition, the peak widths are ~80 Th. The observation of Hb tetramers directly from dried blood spots is an exciting one: the native LESA mass spectrometry approach could be useful in studying disorders of hemoglobin synthesis such as thalassemia. These disorders can result in the production of unusual hemoglobin tetramers such as Hb Barts, a tetramer composed of four gamma chains, or HbH, a tetramer composed of four beta chains [39].Figure 4


Native Liquid Extraction Surface Analysis Mass Spectrometry: Analysis of Noncovalent Protein Complexes Directly from Dried Substrates.

Martin NJ, Griffiths RL, Edwards RL, Cooper HJ - J. Am. Soc. Mass Spectrom. (2015)

Native LESA mass spectra of hemoglobin from dried blood spots: (a) Orbitrap mass analyzer; (b) Q-TOF mass analyzer; (c) ‘contact’ LESA with Q-TOF mass analyzer
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4494149&req=5

Fig4: Native LESA mass spectra of hemoglobin from dried blood spots: (a) Orbitrap mass analyzer; (b) Q-TOF mass analyzer; (c) ‘contact’ LESA with Q-TOF mass analyzer
Mentions: Figure 4 shows the results obtained following native LESA MS of dried blood spots on filter card. The concentration of hemoglobin in human blood is approximately 130 mg/mL for a healthy adult male and 120 mg/mL for a healthy adult female [38]. Results obtained from the Orbitrap (Figure 4a) were similar to those of the hemoglobin standard, showing the presence of (αβ)2H dimers, (αβ)H dimers, αH, βH, and unbound monomers. The relative abundance of the (αβ)2H dimer was greater from the DBS sample than the protein standard. This observation may be the result of reducing the inlet capillary temperature to 200°C. Tetramers were not observed in the Orbitrap data; however, these were observed in the Q-TOF mass spectrum (Figure 4b). Native LESA MS using the Q-TOF resulted in detection of (αβ)24H tetramers, αβ2H dimers, αβH dimers, αH, and βH. As for the standard protein, the tetramer peaks are broad (~100 Th) wide, but the relative abundance of the tetramers is much higher. Figure 4c shows the mass spectrum obtained following ‘contact’ LESA sampling [23] of DBS. The results suggest that this approach is particularly suitable for the analysis of Hb tetramers from DBS. The relative abundance of the peaks corresponding to the tetramers is greater than following standard LESA. In addition, the peak widths are ~80 Th. The observation of Hb tetramers directly from dried blood spots is an exciting one: the native LESA mass spectrometry approach could be useful in studying disorders of hemoglobin synthesis such as thalassemia. These disorders can result in the production of unusual hemoglobin tetramers such as Hb Barts, a tetramer composed of four gamma chains, or HbH, a tetramer composed of four beta chains [39].Figure 4

Bottom Line: Here, we demonstrate native LESA mass spectrometry of noncovalent protein complexes of myoglobin and hemoglobin from a range of surfaces.Heme-bound dimers and monomers were also observed.The 'contact' LESA approach was particularly suitable for the analysis of hemoglobin tetramers from DBS.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

ABSTRACT
Liquid extraction surface analysis (LESA) mass spectrometry is a promising tool for the analysis of intact proteins from biological substrates. Here, we demonstrate native LESA mass spectrometry of noncovalent protein complexes of myoglobin and hemoglobin from a range of surfaces. Holomyoglobin, in which apomyoglobin is noncovalently bound to the prosthetic heme group, was observed following LESA mass spectrometry of myoglobin dried onto glass and polyvinylidene fluoride surfaces. Tetrameric hemoglobin [(αβ)2(4H)] was observed following LESA mass spectrometry of hemoglobin dried onto glass and polyvinylidene fluoride (PVDF) surfaces, and from dried blood spots (DBS) on filter paper. Heme-bound dimers and monomers were also observed. The 'contact' LESA approach was particularly suitable for the analysis of hemoglobin tetramers from DBS.

No MeSH data available.


Related in: MedlinePlus