Impact of sample preparation in peptide/protein profiling in human serum by MALDI-TOF mass spectrometry.
The low molecular weight (LMW) serum proteome (<15 kDa) is the most generally informative from a medical point of view. Different sample pre-treatment approaches and devices for serum depletion in high-abundant proteins were tested in order to analyze, by MALDI-TOF-MS (both in "linear" and "reflectron" acquisition mode), the serum low molecular weight proteins/peptides. The best results in terms of detected ions number and abundance were obtained by using ultrafiltration of serum on 30 kDa molecular weight cut off membranes followed by miniaturized reverse-phase solid-phase extraction (mu-SPE) as sample pre-treatment; this procedure yielded also satisfactory within-sample and sample-to-sample repeatability (on both m/z values and peak intensity of the main observable ions). The procedure was finally applied to serum samples of breast cancer patients, and the relevant results compared to "normal" samples seem to be promising for the individuation of different profiles ("linear" and "reflectron" mode) and/or peptides capable of differentiating for malignancies ("reflectron" mode).
Università degli Studi di Bari, Centro Interdipartimentale di Ricerca S.M.A.R.T., Dipartimento di Chimica, Via Orabona 4, 70126-Bari, Italy.
Antonella
A
Aresta
Cosima D
CD
Calvano
Francesco
F
Palmisano
Carlo G
CG
Zambonin
Alessandro
A
Monaco
Stefania
S
Tommasi
Brunella
B
Pilato
Angelo
A
Paradiso
D015415:Biomarkers / Q000097:blood / Q000737:chemistry / Q000378:metabolism; D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D001943:Breast Neoplasms / Q000097:blood; D005260:Female; D006801:Humans; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D052617:Solid Phase Microextraction / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014462:Ultrafiltration / Q000379:methods
D015415:Biomarkers; D001798:Blood Proteins; D012709:Serum Albumin