Protein extraction from diatom for MALDI analysis

Diatoms is one of the main group of algae that can pro-duce natural silica particles from cell wall, which can be used as a natural sorbent. Biosilica is obtained by remov-ing the organic part from diatom cells. The biosilica parti-cle is strictly defined (e.g. by structure of pores) and pos-sess active functional groups such as carboxyl, amine. For further analysis of organic deposit of biosilica, a MAL-DI analysis was carried out. This method is widely used for a fast peptides/protein analysis as well as microorgan-ism identification.

Aim of the study: Introduction:

Investigation of protein extracted from natural silica col-

lected from diatoms using matrix-assisted laser desorp-

tion/ionization time-of-flight mass spectrometry (MALDI-

TOF-MS) method, alongside with the investigation on

which extraction reagent was the most suitable for this

analysis.

Results and discussion :

Conclusions:

For protein extraction, five different combination of solutions were used: 0,1% ACN, 0,1% TFA, TA30, 5% TFA and 0,1% TFA/ACN solutions were used. For MALDI analysis silica extractions were spotted on MALDI target and analysed with Bruker Ultraflex II TOF/TOF MALDI mass. The analysis of peptides from biosilica showed, that almost all peaks con-tained modified peptides residue or additions to matrix molecules. TA30 extraction spectra has been chosen as it had the most peaks throughout all. Biggest peaks containing amino acids are showed in table.

Acknowledgements:

The experiment has been carried out in the Center for Mod-

ern Interdisciplinary Technologies at Nicolaus Copernicus

University. Scholarships from Erasmus+ programme No.SM-

SMP-2016/17-56 and No.SM-SMP-2016/17-56 is acknowl-

edged.

The results showed that the best solution for extraction

was TA30 as it had the most recognized peaks. Alt-

hough further research has to be carried out for reliabil-

ity of results.

Lina Stepanauskaitė1, Vita Tumosaitė

1, Paweł Pomastowski

2,

Boguslaw Buszewski2,3

, Audrius Maruška1

1Instrumental Analysis Open Access Centre, Faculty of Natural Sciences, Vytautas Magnus University, Vileikos 8, LT-44404 Kaunas, Lithuania

2Centre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University, ul. Gagarina 7, 87-100 Toruń, Poland

3Department of Environmental Chemistry and Bioanalytics, Nicolaus Copernicus University, ul. Gagarina 7, 87-100 Toruń, Poland

Molecular Path m/z

[M+2H+Gln]+ 320.365

[M+H+CO2+Thr]+ 335.362

[M+H+Gly+Thr]+ 348.473

[M+H+Gly+Pnt]+ 379.287

[M+2H+HF+CO2+Trp+ PE]+ 404.472

[M+Asp+Pro+C3H6]+ 443.353

[M+H+Gly+Ala+Tyr]+ 481.140

[M+2H+Gln+Pro+Val]+ 516.208

[M+H+Gly+Ala+Tyr+Ser]+ 568.208

[M+H+Gln+Tyr+Ser+H2O+C2H4]+ 614.105

[M+H+Gly+Ala+Tyr+Ser+PS]+ 672.073

[M+Asp+Pro+HN]+ 766.047

[M+2H+Gly+Ala+

Tyr+Ser+2Na+CH3CN+OAc+PS+Na]+

841.112

Diatoms—

Pseudostaurosira trai-

norii

Isolation of

peptides MALDI

target

Ultraflex/MALDI –

TOF TOF MS

Scheme of workflow

Gly-Ala-Tyr-Ser

Asp-Pro

Gln-Pro-Val

Fig. 2. Spectra of biosilica extracted using TA30 solution

Fig.1 Molecular path of spectra peaks

diatoms