Protein identification by in-gel digestion, high-performance liquid chromatography, and mass spectrometry: peptide analysis by complementary ionization techniques

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Protein identification by in-gel digestion, high-performance liquid chromatography, and mass spectrometry: peptide analysis by complementary ionization techniques. / Medzihradszky, Katalin F.; Leffler, Hakon; Baldwin, Michael A.; Burlingame, A L.

I: Journal of the American Society for Mass Spectrometry, Vol. 12, Nr. 2, 2001, s. 215-221.

Forskningsoutput: TidskriftsbidragArtikel i vetenskaplig tidskrift

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T1 - Protein identification by in-gel digestion, high-performance liquid chromatography, and mass spectrometry: peptide analysis by complementary ionization techniques

AU - Medzihradszky, Katalin F.

AU - Leffler, Hakon

AU - Baldwin, Michael A.

AU - Burlingame, A L

PY - 2001

Y1 - 2001

N2 - A biologically active protein fraction was isolated from rabbit intestine, purified by one-dimensional SDS-PAGE and stained with Coomassie Brilliant Blue. A predominant band of approximately 110-130 kDa was excised and digested in-gel with trypsin. The resulting peptides were extracted then separated by microbore reversed-phase high-performance liquid chromatography (HPLC). Mass spectrometric data from one HPLC fraction obtained by two different ionization techniques proved to be complementary. Matrix-assisted laser desorption/ionization (MALDI) showed nine peptide masses, which by post source decay analysis and database searching were attributed to two proteins. Nanoflow electrospray analysis performed on a hybrid tandem mass spectrometer of quadrupole-quadrupole-orthogonal acceleration time-of-flight (QqTOF) geometry detected six additional peptide components. On the basis of the additional peptides and superior quality collision-induced dissociation spectra typical of this instrument type, two further proteins were identified. The resolution afforded by the QqTOF instrument permitted charge state determination for the fragment ions while preserving the high detection sensitivity that was essential in obtaining the composition of this mixture of proteins.

AB - A biologically active protein fraction was isolated from rabbit intestine, purified by one-dimensional SDS-PAGE and stained with Coomassie Brilliant Blue. A predominant band of approximately 110-130 kDa was excised and digested in-gel with trypsin. The resulting peptides were extracted then separated by microbore reversed-phase high-performance liquid chromatography (HPLC). Mass spectrometric data from one HPLC fraction obtained by two different ionization techniques proved to be complementary. Matrix-assisted laser desorption/ionization (MALDI) showed nine peptide masses, which by post source decay analysis and database searching were attributed to two proteins. Nanoflow electrospray analysis performed on a hybrid tandem mass spectrometer of quadrupole-quadrupole-orthogonal acceleration time-of-flight (QqTOF) geometry detected six additional peptide components. On the basis of the additional peptides and superior quality collision-induced dissociation spectra typical of this instrument type, two further proteins were identified. The resolution afforded by the QqTOF instrument permitted charge state determination for the fragment ions while preserving the high detection sensitivity that was essential in obtaining the composition of this mixture of proteins.

U2 - 10.1016/S1044-0305(00)00214-2

DO - 10.1016/S1044-0305(00)00214-2

M3 - Article

VL - 12

SP - 215

EP - 221

JO - Journal of the American Society for Mass Spectrometry

JF - Journal of the American Society for Mass Spectrometry

SN - 1879-1123

IS - 2

ER -