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In order to further define the structural repertoire of PSA and PAP glycans, permethylation28

of the PNGaseF released glycans was done for each clinical cohort, followed by MALDI-
TOF/TOF analysis. Because of the broad diversity of the indicated structural classes from the
HPLC analyses, the goal was not to identify disease specific changes, but to better define all
of the possible glycan structures detected on seminal plasma PSA and PAP.
Permethylation of glycans aids in the stability of the terminal sialylated residues for detection
by MALDI-TOF/TOF, and simplifies the spectra to primarily Na+ adducts, allowing for less
complex annotation of mass peaks. From the permethylation experiments, we were able to
detect multiple glycoforms for both PAP and PSA (Tables 1 and 2). A representative
MALDITOF spectra of PAP derived permethylated glycans from prostate cancer samples are
shown in Figure 7. Cumulatively, 21 structural classes of PAP glycans were detected
representing primarily high-mannose and complex subtypes, with a few potential hybrid sub-
types represented as well (Table 1). Permethylated glycans cleaved from PSA seemed to be
mostly bi- and tri-antennary structures of the complex sub-type, but represented 40 potential
classes, including high mannose and hybrid sub-types detected as well (Table 2). These
results are consistent with structures previously reported for PSA glycans from serum and
seminal fluids17–19, 2

Untuk lebih menentukan repertoar struktural PSA dan PAP glycans, permethylation28 dari
PNGaseF dirilis glycans dilakukan untuk setiap kohort klinis, diikuti oleh analisis MALDI-
TOF / TOF. Karena keragaman yang luas dari kelas struktural yang ditunjukkan dari analisis
HPLC, tujuannya bukan untuk mengidentifikasi perubahan spesifik penyakit, tetapi untuk
lebih mendefinisikan semua struktur glycan yang mungkin terdeteksi pada PSA dan PAP
plasma seminalis.
Permethylation of glycans membantu stabilitas residu sialylated terminal untuk deteksi oleh
MALDI-TOF / TOF, dan menyederhanakan spektrum untuk terutama Na + adduct,
memungkinkan untuk lebih sedikit anotasi massa puncak. Dari percobaan permethylation,
kami mampu
mendeteksi beberapa glikoform untuk PAP dan PSA (Tabel 1 dan 2). Sebuah spektrum
MALDITOF representatif dari PAP diturunkan glycans permethylated dari sampel kanker
prostat ditunjukkan pada Gambar 7. Secara kumulatif, 21 kelas struktural PAP glycans
terdeteksi mewakili terutama subtipe mannose dan kompleks yang tinggi, dengan beberapa
sub-jenis hibrida potensial diwakili juga ( Tabel 1). Glycans permethylated dibelah dari PSA
tampaknya sebagian besar struktur bi-dan tri-antennary dari sub-jenis kompleks, tetapi
mewakili 40 kelas potensial, termasuk mannose tinggi dan hibrida sub-jenis terdeteksi juga
(Tabel 2). Hasil ini konsisten dengan struktur yang sebelumnya dilaporkan untuk glycans
PSA dari serum dan cairan mani17-19, 2

MALDI is achieved in two steps. In the first step, the compound to be analysed is dissolved
in a solvent containing in solution small organicmolecules, called the matrix. Thesemolecules
must have a strong absorption at the laser wavelength. This mixture is dried before analysis
and any liquid solvent used in the preparation of the solution is removed. The result is a ‘solid
solution’ deposit of analyte-doped matrix crystals. The analyte molecules are embedded
throughout the matrix so that they are completely isolated from one another. The second step
occurs under vacuum conditions inside the source of the mass spectrometer. This step
involves ablation of bulk portions of this solid solution by intense laser pulses over a short
duration. The exact mechanism of theMALDI process is not completely elucidated [24,25].
However, irradiation by the laser induces rapid heating of the crystals by the accumulation of
a large amount of energy in the condensed phase through excitation of the matrix molecules.
The rapid heating causes localized sublimation of the matrix crystals, ablation of a portion of
the crystal surface and expansion of the matrix into the gas phase, entraining intact analyte in
the expanding matrix plume.
Ionization reactions can occur under vacuum conditions at any time during this process but
the origin of ions produced in MALDI is still not fully understood [27, 28]. Among the
chemical and physical ionization pathways suggested for MALDI are gas-phase
photoionization, excited state proton transfer, ion–molecule reactions, desorption of
preformed ions, and so on. The most widely accepted ion formation mechanism involves
proton transfer in the solid phase before desorption or gas-phase proton transfer in the
expanding plume from photoionized matrix molecules. The ions in the gas phase are then
accelerated by an electrostatic field towards the analyser. Figure 1.15 shows a diagram of the
MALDI desorption ionization process. MALDI is more sensitive than other laser ionization
techniques. Indeed, the number of matrix molecules exceeds widely those of the analyte, thus
separating the analytemolecules and thereby preventing the formation of sample clusters that
inhibit the appearance of molecular ions. The matrix also minimizes sample damage from the
laser pulse by absorbing most of the incident energy and increases the efficiency of energy
transfer from the laser to the analyte. So the sensitivity is also highly increased. MALDI is
also more universal than the other laser ionization techniques. Indeed, it is not necessary to
adjust the wavelength to match the absorption frequency of each analyte because it is the
matrix that absorbs the laser pulse. Furthermore, because the process is independent of the
absorption properties and size of the compound to be analysed, MALDI allows the desorption
and ionization of analytes with very high molecular mass in excess of 100 000 Da. For
example, MALDI allows the detection of femtomoles of proteins with molecular mass up to
300 000 Da.
MALDI mass spectrometry has become a powerful analytical tool for both synthetic
polymers and biopolymers. Typical MALDI spectra include mainly the monocharged
molecular species by protonation in positive ion mode. More easily deprotonated compounds
are usually detected in negative ion mode. Some multiply charged ions, some

The use of MALDI to image biological materials is another interesting application. Indeed, as
with LD and SIMS, MALDI has been used to map the distribution of targeted biomolecules
in tissue. It allows for example the study of peptides, proteins and other biomolecules directly
on tissue sections. Contrary to most other ionization sources that yield a continuous ion beam,
MALDI is a pulsed ionization technique that produces ions in bundles by an intermittent
process. The pulsed nature of the MALDI source is well suited for the time of-flight (TOF)
analyser. In addition, the TOF analyser has the ability to analyse ions over a wide mass range
and thus can analyse the high-mass ions generated by MALDI. Altogether, this explains why
most MALDI spectra have been obtained with MALDI-TOF spectrometers. However, there
is no fundamental reason to limit the use of MALDI sources with TOF analysers. MALDI
sources have also been coupled to other mass analysers, such as ion trap or Fourier transform
mass spectrometers. These instruments allowMS/MS analysis to be performed much more
powerfuly and easier realized than using TOF instruments. Furthermore, Fourier transform
mass spectrometers reach high resolutions