New desorption strategies for the mass spectrometric analysis of macromolecules

Abstract

We present two new desorption strategies for the mass spectrometric analysis of macromolecules. These desorption strategies are based on the molecular design and construction of two general classes of Sample 'probe' surfaces. The first class of surfaces is designed to enhance the desorption of intact macromolecules presented alone (neat) to the surface; we call this surface-enhanced neat desorption (SEND). The availability of probe surfaces derivatized with, or composed of, multiple types and defined numbers of energy-absorbing molecules will facilitate investigations of energy transfer and desorption/ionization mechanisms. The second class of probe surfaces is designed to enhance the desorption of specific macromolecules captured directly from unfractionated biological fluids and extracts; we call this surface-enhanced affinity capture (SEAC). Use of these new probe surfaces as chemical& defined solid-phase reaction centers will facilitate protein discovery through molecular recognition in situ, and also macromolecular structure analysis through the sequential chemical and/or enzymatic modification of the adsorbed analyte in situ. Specific examples of laser-assisted SEND and SEAC timeof-flight mass spectrometry are presented to illustrate the potential for increased selectivity, analyte detection sensitivity, and mass measurement accuracy.The introduction of matrix molecules to assist the laser-induced desorption/ionization (MALDI) of intact macromolecules has enabled relatively small quantities of large nonvolatile biopolymers to be evaluated by time-of-flight (TOF) mass spectrometry.' Many new types of investigations have been enabled by this technology.* The ability to measure precise differences in molecular mass enables problems associated with unknown protein structures to be solved more r e a d i l~.~.~ Thus, the future of biological mass spectrometry will be defined by simpler and more sensitive means to monitor specific changes in protein and peptide mass.Our laboratory has developed MALDI-TOF to explore various specific ligand-ligate interactions and to analyze protein structure;%" both approaches have been based largely on biochemical reactions performed directly on the probe surface (i.e., in situ) to exploit the availability of analyte not consumed by previous laserinduced desorption events. However, the required presence of a large molar excess of acidic matrix and the random deposition of embedded macromolecular analytes in and around an undefined array of matrix crystals has limited the use of the probe surface as an effective stationary phase from which to conduct, by differential mass spectrometry, a sequential in situ approach to the investigation of protein structure and function.An effective mass spectrometric strategy for protein structure analysis may be realized if each of several diagnostic alterations in mass can be brought about as a result of controlled chemical and/or enzymatic modification events performed directly and sequentially in situ, i.e., directly on the probe surface. In fa...

CHCA-modified Au nanoparticles for laser desorption ionization mass spectrometric analysis of peptides

“…UV-absorbing molecules, such as CHCA [29,30], 2,5-dihydroxybenzoic acid [31,32], and selfassembled monolayer reagents such as methyl N-(4-mercaptophenyl)carbamate [33], have been immobilized on the solid support surface to assist the desorption ionization of samples in SALDI-MS. Presence of a proton donor and high extinction coefficients at laser exciting wavelengths are crucial for MALDI [34] and SALDI-MS [22,35].…”

Analysis of peptides and proteins affinity-bound to iron oxide nanoparticles by MALDI MS

“…However, the sensitivity improvement was limited by the number of binding sites on the target. In another approach, the so-called surface-enhanced affinity capture (SEAC), micrometer-sized beads made for chromatography column were used to capture peptides and proteins from sample solution [16]. Various types of beads have been used, which include reverse-phase chromatographic beads [17,18] and immobilized metal ion beads [19 -21].…”

Epitope mapping of the gastrin‐releasing peptide/anti‐bombesin monoclonal antibody complex by proteolysis followed by matrix‐assisted laser desorption ionization mass spectrometry

“…Recently, MALDI/TOF has been used to analyze a protein directly attached to an affinity matrix. The investigators named this technique surface-enhanced affinity capture (SEAC) time-of-flight mass spectrometry (Hutchens &Yip, 1993). Human lactoferrin from urine was captured with immobilized single-stranded DNA and analyzed directly by MALDI/TOF (Hutchens & Yip, 1993).…”

“…The investigators named this technique surface-enhanced affinity capture (SEAC) time-of-flight mass spectrometry (Hutchens &Yip, 1993). Human lactoferrin from urine was captured with immobilized single-stranded DNA and analyzed directly by MALDI/TOF (Hutchens & Yip, 1993). We presumed that the direct analysis of an affinity-bound analyte could be accomplished with an immobilized antibody as the affinity capture device.…”