Although the cell line K562 reportedly expresses a single species of β1 integrin, α5β1, surface staining with monoclonal antibodies JB1A, 12G10 and B3B11 to the β1 chain clearly demonstrated differences in the expression levels of the epitopes detected by these antibodies. The present studies were initiated to determine the basis for this molecular heterogeneity in the integrins. Cross-linking of surface integrins with B3B11 caused their selective aggregation. This distribution was similar to that observed for the α5 chain. In contrast, cross-linking the β1 chains with 12G10 did not cause codistribution of α5, suggesting that these two species were not associated on the cell surface. Immunoprecipitates of the surface integrins of K562 cells indicated the presence of 120 and 140 kDa forms of the β1 chain which were detected by 12G10 and B3B11, respectively. Immunological, biochemical and mass spectrometric analysis of K562 surface integrins also failed to demonstrate the presence of any α chain in association with the 120 kDa species of β1 of K562 cells. Treatment of the two forms of β1 with PGNase reduced their masses to ∼90 kDa, suggesting that N-glycosylation was responsible for the mass differences. Collectively, these results provide evidence for a novel species of β1 on the cell surface, which does not appear to be associated with any α chain. The data also suggest that differences in glycosylation may be involved in defining the association between the integrin α and β chains and the functional properties of these integrins.

The adhesion blocking antibody 3S3 was used to probe the regulation of alpha5beta1 integrin mediated adhesion in K562 cells. This antibody prevented cellular adherence but it did not interfere with ligand binding by cells or purified integrin. Interaction with 3S3 induced change in the cytoskeletal organization resulting in extensive filopodia formation. The antibody also prevented ligand and anti-integrin antibody induced phosphorylation of FAK in a trans acting fashion. MS based analysis of 3S3 induced integrin containing complexes identified rasGAP SH3 binding protein 1, G3BP1, as a component of these structures. The G3BP1 binding molecule, rasGap(120), was also identified in the complexes. Microscopic examination confirmed the recruitment of a component of cellular G3BP1 and rasGap(120) pools to sites of integrin cross-linking. G3BP1 was also observed in the 3S3 induced filopodia. In untreated cells, G3BP1 was shown to associate with submembranous regions involved in cellular polarization. Collectively, these results suggest that G3BP1 and rasGap(120) can be recruited to sites of integrin ligation where they may play a role in cytoskeletal reorganization. Such changes may result in reduced adhesive potential and account for the 3S3 effects on cellular adhesion. It should be emphasized that these results do not necessarily indicate a direct interaction of integrin with G3BP1 and rasGap(120).

The determination of monoclonal antibody specificity is dependent upon the availability of purified antigen. Such material is not always available and this has proven to be one of the rate-limiting steps in monoclonal antibody production. The aim of the present study was to develop a generic approach to defining antibody specificity that bypassed the need for pure antigens through the use of proteomics. The scheme and its application to several biological mixtures are described. The results demonstrate the ability of the approach to identify antibodies against both the major components and the minor contaminants of a protein mixture. This approach should markedly enhance the characterization of antibodies to complex antigen mixtures.

Tandem instruments, such as triple-quadrupole or quadrupole-time-of-flight mass spectrometers, often use collisional damping ion guides for the purpose of cooling and focusing the primary ion beam, or as a collision cell for tandem mass spectrometry experiments. A small, axial field to reduce the ion residence time in such devices can give considerable improvements in performance. Reduction of the residence time reduces adduct formation caused by unwanted gas-phase reactions and allows multiple reaction monitoring (MRM) and some other types of scan to be carried out more rapidly without the risk of reaction cross-talk. Here we propose a novel electrode arrangement for creation of a suitable axial field in a multipole ion guide without significantly reducing the m/z window. As an example, a second set of four electrodes is added to an existing quadrupole collision cell. The same DC potential is applied to all four extra electrodes, which are shaped in the longitudinal direction to create a suitable axial field inside the device. The potential of these electrodes and the DC bias of the main rods determine the axial field strength. The idea was tested on a modified quadrupole collision cell of a MALDI-QqTOF instrument and experimental results are presented here, as well as a simplified theory of operation.

In a previous paper, the observation of spontaneous desorption (SD) of negative ions in a time-of-flight mass spectrometer has been reported. The ion emission was found to depend on a high potential difference between the target and the ion accelerating grid placed a few mm apart from it. This paper shows that the SD of ions is a secondary ion emission phenomenon in which charged particles coming from the grid surface hit the target. The incident particles are either directly extracted from the grid by the applied high electric field and/or are secondary ions of impinging charged particles generated at the sample layer surface, again by a field-induced process. Measurements of the ion multiplicity suggest multiatomic ions as incident particles. The SD effect could also be established for positive ions.

We have studied the erosion rates of icy films under irradiation by a wide variety of ion beams, from H to Kr, and energies from 1 keV to 100 MeV. The films are prepared by condensation of vapors (H2O, CO2, NH3 and mixtures) onto KBr substrates maintained at liquid nitrogen temperature. The disappearance of H2O has been measured by on line Fourier transform infrared spectometry. The yields have been found to depend upon the properties of the beam as well as the thickness of the films, in both the keV and the MeV ranges. In the latter, values as high as 105 H2O molecules/incident Kr ion have been observed, for 1.5 μm thick films. The linear dependence of the yields on film thickness is consistent with a mechanism operating all along the ion tracks, by which molecules are either directly ejected or chemically transformed.

The heavy ion linear accelerator of the Institut de Physique Nucleaire has been used to study the influence of the projectile charge state q i on secondary ion emission. Ions of Ne, Ar or Kr with a velocity of 1.16 MeV/u bombarded thin films of organic and inorganic solids. The experimental arrangement is described. The ion emission yield is strongly dependent on the charge state of the incident ion qi and of its atomic number (nature of the projectile). The emission yield between the three types of projectiles varies as q4eq where q eq is the equilibrium charge state within the material for each projectile.

The ${\mathrm{H}}^{+}$ secondary-ion yield from the surface of solid targets of Au, C, and nitrocellulose has been used to measure mean charges of energetic ions of $^{84}\mathrm{Kr}$ and $^{40}\mathrm{Ar}$ (at 1.16 MeV/u) at the beam-exit surface. Values for Kr ions were found to be 1.6 to 3 electronic charges ($e$) less than those found 70 ns later, after Auger decay. No such difference was observed for Ar ions of the same velocity.

Secondary ions ${[{(\mathrm{CsI})}_{n}\mathrm{Cs}]}^{+}$, with $n$ up to \ensuremath{\sim} 40, were produced by 8-keV ${\mathrm{Cs}}^{+}$ bombardment of CsI. The yield of clusters decreased smoothly with $n$ when observed in a time-of-flight mass spectrometer at effective times \ensuremath{\sim} 0.2 \ensuremath{\mu}s after emission. Clusters with $n>7$ were found to be metastable, with lifetimes \ensuremath{\ll} 100 \ensuremath{\mu}s. A large anomaly in the population of the disintegration products was measured at \ensuremath{\sim} 70 \ensuremath{\mu}s after emission, $n=13$ clusters being favored and $n=14 \mathrm{and} 15$ suppressed.