42
L. CASTELLANOS-SERRA et al.
allows the in-solution-like conditions for digestion, with shorter digestion times
and a rapid procedure for peptide recovery. The shorter digestion times also have
the advantage of reducing non-specific cleavages and protease auto-proteolysis,
while increasing the chance for obtaining longer pep tides that facilitate their
analysis. There are neither artifact peaks from the staining dye in the chromatogram nor CB-protein adducts detected in MS analysis.
2.10
Sample Preparation for Analysis by MALDI MS
The solution containing the eluted pep tides is concentrated under vacuum to a
volume of about 5-10 !ll. Pep tides (about 0.5 !ll) are analyzed on dihydroxybenzoic acid spotted on the probe tips of the spectrometer. In cases where the salt
content of the sample interferes with an adequate ionization of the pep tides,
microdesalting is accomplished by using a commercial micro reversed phase column (ZipTip; Millipore). Alternatively, the peptide sample can be desalted on the
MALDI probe tip, by spotting it on a nitrocellulose/alpha-cyano-4-hydroxytrans-cinnamic acid matrix, and rinsing several times with water after the analyte solution has dried completely, as described by Jensen et al. (1997)
2.11
Sample Preparation for Analysis by ESI-MS/MS (Reid et ai, 1998; lugaro et ai, 1998)
The solution containing the eluted peptides is concentrated under vacuum centrifugation to a volume of about 40-50 !ll. Peptides are analyzed by directly loading the digest solution onto the reversed-phase capillary HPLC column connected to the electrospray Ion-trap mass spectrometer. This is accomplished by
loading the sample (25-50 !ll/Inj) at a higher flow rate of 5 -10 !ll/min onto a 0.2mm LD. column in primary Buffer A (0.1 % v/v TFA). Once completed, the flow
rate is reset to the operating flow rate of 1-1.5 !lllmin. In this case, the salt content of the sample does not interfere with the ionization of the peptides, as microdesalting and buffer exchange is accomplished during loading onto the micro
reversed phase columns.
2.12
Preparation of Capillary Reversed-Phase Microcolumns
Procedure (Moritz & Simpson, 1992; Moritz et ai, 1996a)
The following step by step procedure for constructing fused-silica micro columns
is illustrated using the construction of a 150 x 0.2-mm LD. column. First, two
lengths of fused-silica tubing need to be cut to form the column body (0.20-mm
LD. x 0.32-mm O.D. x 150mm) and the column fluid transfer line (0.05-mm
LD. x 0.19-mm O.D. x 200mm) with the use of a fused-silica sapphire knife. Next,
cut out a column frit, using the column body fused-silica tubing as a punch, from
a disk of 0.45!lm porosity hydrophilic PVDF (Millipore, cat. HVLP04700) and
insert it approximately 5 mm into the column body using the fused-silica exiting
tubing. The micro column body and exiting tubing are then permanently posi-
L. CASTELLANOS-SERRA et al.
allows the in-solution-like conditions for digestion, with shorter digestion times
and a rapid procedure for peptide recovery. The shorter digestion times also have
the advantage of reducing non-specific cleavages and protease auto-proteolysis,
while increasing the chance for obtaining longer pep tides that facilitate their
analysis. There are neither artifact peaks from the staining dye in the chromatogram nor CB-protein adducts detected in MS analysis.
2.10
Sample Preparation for Analysis by MALDI MS
The solution containing the eluted pep tides is concentrated under vacuum to a
volume of about 5-10 !ll. Pep tides (about 0.5 !ll) are analyzed on dihydroxybenzoic acid spotted on the probe tips of the spectrometer. In cases where the salt
content of the sample interferes with an adequate ionization of the pep tides,
microdesalting is accomplished by using a commercial micro reversed phase column (ZipTip; Millipore). Alternatively, the peptide sample can be desalted on the
MALDI probe tip, by spotting it on a nitrocellulose/alpha-cyano-4-hydroxytrans-cinnamic acid matrix, and rinsing several times with water after the analyte solution has dried completely, as described by Jensen et al. (1997)
2.11
Sample Preparation for Analysis by ESI-MS/MS (Reid et ai, 1998; lugaro et ai, 1998)
The solution containing the eluted peptides is concentrated under vacuum centrifugation to a volume of about 40-50 !ll. Peptides are analyzed by directly loading the digest solution onto the reversed-phase capillary HPLC column connected to the electrospray Ion-trap mass spectrometer. This is accomplished by
loading the sample (25-50 !ll/Inj) at a higher flow rate of 5 -10 !ll/min onto a 0.2mm LD. column in primary Buffer A (0.1 % v/v TFA). Once completed, the flow
rate is reset to the operating flow rate of 1-1.5 !lllmin. In this case, the salt content of the sample does not interfere with the ionization of the peptides, as microdesalting and buffer exchange is accomplished during loading onto the micro
reversed phase columns.
2.12
Preparation of Capillary Reversed-Phase Microcolumns
Procedure (Moritz & Simpson, 1992; Moritz et ai, 1996a)
The following step by step procedure for constructing fused-silica micro columns
is illustrated using the construction of a 150 x 0.2-mm LD. column. First, two
lengths of fused-silica tubing need to be cut to form the column body (0.20-mm
LD. x 0.32-mm O.D. x 150mm) and the column fluid transfer line (0.05-mm
LD. x 0.19-mm O.D. x 200mm) with the use of a fused-silica sapphire knife. Next,
cut out a column frit, using the column body fused-silica tubing as a punch, from
a disk of 0.45!lm porosity hydrophilic PVDF (Millipore, cat. HVLP04700) and
insert it approximately 5 mm into the column body using the fused-silica exiting
tubing. The micro column body and exiting tubing are then permanently posi-