Details

Autor(en) / Beteiligte

• Möhring, Thomas
• Kellmann, Markus
• Jürgens, Michael
• Schrader, Michael

Titel

Top-down identification of endogenous peptides up to 9 kDa in cerebrospinal fluid and brain tissue by nanoelectrospray quadrupole time-of-flight tandem mass spectrometry

Ist Teil von

• Journal of mass spectrometry., 2005-02, Vol.40 (2), p.214-226

Ort / Verlag

Chichester, UK: John Wiley & Sons, Ltd

Erscheinungsjahr

2005

Quelle

MEDLINE

Beschreibungen/Notizen

• Recent work on protein and peptide biomarker patterns revealed the difficulties in identifying their molecular components, which is indispensable for validation of the biological context. Cerebrospinal fluid and brain tissue are used as sources to discover new biomarkers, e.g. for neurodegenerative diseases. Many of these biomarker candidates are peptides with a molecular mass of <10 kDa. Their identification is favourably achieved with a ‘top‐down’ approach, because this requires less purification and an enzymatic cleavage will often not yield enough specific fragments for successful database searches. Here, we describe an approach using quadrupole time‐of‐flight mass spectrometry (TOFMS) as a highly efficient mass spectrometric purification and identification tool after off‐line decomplexation of biological samples by liquid chromatography. After initial peptidomic screening with matrix‐assisted laser desorption/ionization (MALDI) TOFMS, the elution behaviour in chromatography and the exact molecular mass were used to locate the same signals in nanoelectrospray measurements. Most of the peaks detected in MALDI‐TOFMS could be retrieved in nanoelectrospray quadrupole TOFMS. Suitable collision energies for informative fragment spectra were investigated for different parent ions, charge states and molecular masses. After collision‐induced dissociation, the resulting fragmentation data of multiply charged ions can become much more complicated than those derived from tryptic peptide digests. However, the mass accuracy and resolution of quadrupole TOF instruments results in high‐quality data suitable for determining peptide sequences. The protein precursor, proteolytic processing and post‐translational modifications were identified by automated database searches. This is demonstrated by the exemplary identifications of thymosin beta‐4 (5.0 kDa) and NPY (4.3 kDa) from rat hypothalamic tissue and ubiquitin (8.6 kDa) from human cerebrospinal fluid. The high data quality should also allow for de novo identification. This methodology is generally applicable for peptides up to a molecular mass of about 10 kDa from body fluids, tissues or other biological sources. Copyright © 2005 John Wiley & Sons, Ltd.