Abstract
In the analysis of a neuroblastoma xenograft implanted in mice using two-dimensional maps, some 85 proteins were found to be up- or down-regulated (out of a total of 264 detected by a medium-sensitivity colloidal Coomassie stain). When these spots were eluted and analysed by mass spectrometry in a quadrupole time of flight mass spectrometer, a number of spots were found to be envelopes of different polypeptide chains. Out of a total of 74 proteins identified, 52 (71%) were found to be singlets, 14 (19%) were doublets, 6 (8%) were triplets, 1 was a quadruplet and 1 a quintuplet. Analysis of the ΔpI and ΔMr of all species contained in a single gel segment eluted helped point out potential errors in protein identification. This was a unique case, in that very minute bioptic sample loads were applied to the gel. In normal cases, where sample loads of ca. 1 mg of total protein are applied and typically at least 1000 spots are visualised, the singlets will be the minority, rarely exceeding 30% of all spots analysed. The experimental data on the abundance of overlapping spots were in excellent agreement with theoretical data calculated on the basis of the statistical theory of spot overlapping, originally proposed by Davis and further developed by some of the authors. Ways and means for minimizing spot overlap and visualising a greater number of spots in a two-dimensional map are discussed.
| Original language | English |
|---|---|
| Pages (from-to) | 2385-2395 |
| Number of pages | 11 |
| Journal | Proteomics |
| Volume | 5 |
| Issue number | 9 |
| DOIs | |
| Publication status | Published - Jun 2005 |
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Keywords
- Neuroblastoma
- Spot overlap
- Two-dimensional maps
ASJC Scopus subject areas
- Genetics
- Molecular Biology
Cite this
Spot overlapping in two-dimensional maps : A serious problem ignored for much too long. / Campostrini, Natascia; Areces, Liliana B.; Rappsilber, Juri; Pietrogrande, Maria Chiara; Dondi, Francesco; Pastorino, Fabio; Ponzoni, Mirco; Righetti, Pier Giorgio.
In: Proteomics, Vol. 5, No. 9, 06.2005, p. 2385-2395.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Spot overlapping in two-dimensional maps
T2 - A serious problem ignored for much too long
AU - Campostrini, Natascia
AU - Areces, Liliana B.
AU - Rappsilber, Juri
AU - Pietrogrande, Maria Chiara
AU - Dondi, Francesco
AU - Pastorino, Fabio
AU - Ponzoni, Mirco
AU - Righetti, Pier Giorgio
PY - 2005/6
Y1 - 2005/6
N2 - In the analysis of a neuroblastoma xenograft implanted in mice using two-dimensional maps, some 85 proteins were found to be up- or down-regulated (out of a total of 264 detected by a medium-sensitivity colloidal Coomassie stain). When these spots were eluted and analysed by mass spectrometry in a quadrupole time of flight mass spectrometer, a number of spots were found to be envelopes of different polypeptide chains. Out of a total of 74 proteins identified, 52 (71%) were found to be singlets, 14 (19%) were doublets, 6 (8%) were triplets, 1 was a quadruplet and 1 a quintuplet. Analysis of the ΔpI and ΔMr of all species contained in a single gel segment eluted helped point out potential errors in protein identification. This was a unique case, in that very minute bioptic sample loads were applied to the gel. In normal cases, where sample loads of ca. 1 mg of total protein are applied and typically at least 1000 spots are visualised, the singlets will be the minority, rarely exceeding 30% of all spots analysed. The experimental data on the abundance of overlapping spots were in excellent agreement with theoretical data calculated on the basis of the statistical theory of spot overlapping, originally proposed by Davis and further developed by some of the authors. Ways and means for minimizing spot overlap and visualising a greater number of spots in a two-dimensional map are discussed.
AB - In the analysis of a neuroblastoma xenograft implanted in mice using two-dimensional maps, some 85 proteins were found to be up- or down-regulated (out of a total of 264 detected by a medium-sensitivity colloidal Coomassie stain). When these spots were eluted and analysed by mass spectrometry in a quadrupole time of flight mass spectrometer, a number of spots were found to be envelopes of different polypeptide chains. Out of a total of 74 proteins identified, 52 (71%) were found to be singlets, 14 (19%) were doublets, 6 (8%) were triplets, 1 was a quadruplet and 1 a quintuplet. Analysis of the ΔpI and ΔMr of all species contained in a single gel segment eluted helped point out potential errors in protein identification. This was a unique case, in that very minute bioptic sample loads were applied to the gel. In normal cases, where sample loads of ca. 1 mg of total protein are applied and typically at least 1000 spots are visualised, the singlets will be the minority, rarely exceeding 30% of all spots analysed. The experimental data on the abundance of overlapping spots were in excellent agreement with theoretical data calculated on the basis of the statistical theory of spot overlapping, originally proposed by Davis and further developed by some of the authors. Ways and means for minimizing spot overlap and visualising a greater number of spots in a two-dimensional map are discussed.
KW - Neuroblastoma
KW - Spot overlap
KW - Two-dimensional maps
UR - http://www.scopus.com/inward/record.url?scp=20644441336&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=20644441336&partnerID=8YFLogxK
U2 - 10.1002/pmic.200401253
DO - 10.1002/pmic.200401253
M3 - Article
C2 - 15880804
AN - SCOPUS:20644441336
VL - 5
SP - 2385
EP - 2395
JO - Proteomics
JF - Proteomics
SN - 1615-9853
IS - 9
ER -