Biology Reference
In-Depth Information
12. The general appearance of the spectra should be checked.
Typically, the majority of tryptic peptides have masses of
~1,500-2,000 Da and appear in the spectrum as a Gaussian-like
distribution of intensities centered around m / z ~1,500. Lots of
larger peaks may indicate incomplete digestion, whereas many
smaller peaks may indicate too long incubation time (leading to
nonspecifi c cleavage), especially if many trypsin autolysis peaks
can also be seen. In addition to the incubation time, the con-
centration of ammonium bicarbonate in the buffer represents
another parameter that can be optimized for particular tissue
types. Trypsin operates at a slightly alkaline pH optimum, so for
samples with a low pH, a higher concentration of ammonium
bicarbonate (up to 80 mM) can be helpful.
13. It may be helpful to cover at least a part of the analyzed tissue
with a coverslip before the application of trypsin. The coverslip
should be removed before matrix application. This area can be
measured as an internal control to evaluate the success of the
digestion.
14. The primary choice of matrix for the measurement of proteins
is usually SA, which usually provides good spectra quality for
proteins of up to 25 kDa even from complex mixtures such as
tissue samples. The main drawback is the size of matrix crys-
tals, which is commonly in the range of ~50
m and limits the
spatial resolution of the MALDI image. If higher spatial reso-
lution is desired, HCCA matrix can be used instead, which
forms smaller crystals. Spatial resolution of up to 20
μ
μ
m for
MSI of proteins has been achieved using HCCA [ 16 ].
References
1. Caprioli RM, Farmer TB, Gile J et al (1997)
Molecular imaging of biological samples: local-
ization of peptides and proteins using MALDI-
TOF MS. Anal Chem 69:4751-4760
2. Rohner TC, Staab D, Stoeckli M et al (2005)
MALDI mass spectrometric imaging of bio-
logical tissue sections. Mech Ageing Dev 126:
177-185
3. Burnum KE, Kristin E, Frappier SL et al (2008)
Matrix-assisted laser desorption/ionization
imaging mass spectrometry for the investiga-
tion of proteins and peptides. Annu Rev Anal
Chem 1:689-705
4. van Hove ERA, Smith DF, Heeren RMA et al
(2010) A concise review of mass spectrometry
imaging. J Chromatogr A 1217:3946-3954
5. Yanagisawa K, Shyr Y, Xu BGJ et al (2003)
Proteomic patterns of tumour subsets in non-
small-cell lung cancer. Lancet 362:433-439
6. Schwartz SA, Weil RJ, Johnson MD et al
(2004) Protein profi ling in brain tumors using
mass spectrometry: Feasibility of a new tech-
nique for the analysis of protein expression.
Clin Cancer Res 10:981-987
7. Cornett DS, Mobley JA, Dias EC et al (2006)
A novel histology-directed strategy for
MALDI-MS tissue profi ling that improves
throughput and cellular specifi city in human
breast cancer. Mol Cell Proteomics 5:
1975-1983
8. Reyzer ML, Hsieh YS, Ng K et al (2003) Direct
analysis of drug candidates in tissue by matrix-
assisted laser desorption/ionization mass spec-
trometry. J Mass Spectrom 38:1081-1092
9. Holscher D, Shroff R, Knop K et al (2009)
Matrix-free UV-laser desorption/ionization
(LDI) mass spectrometric imaging at the
single-cell level: distribution of secondary
Search WWH ::




Custom Search