Gas-Phase Separations of Electrosprayed Peptide Libraries

Self Cite

Recent improvements in ion mobility/time-of-flight mass spectrometry techniques have made it possible to incorporate nano-flow liquid chromatography and collision induced dissociation techniques. This combination of approaches provides a new strategy for detailed characterization of complex systems-such as, combinatorial libraries. With such complex systems, however, comes a tradeoff in the level of detail to which the individual components are characterized. Specifically, if a synthetic step fails unexpectedly, the purity of an entire class of desired molecules may be compromised. With this in mind, a number of strategies for characterizing complex libraries are being developed [2]. Among the most powerful of these is the combination of separation techniques such as liquid chromatography (LC) with mass spectrometry (MS). The advent of electrospray ionization (ESI) [3] allows LC to be coupled with MS in an online fashion. The LC separation simplifies the complex mixture and MS can be used for identification; that is, it is possible to select an initial precursor ion and utilize collision induced dissociation (CID) data to obtain structural information about individual components-an overall LC-MS/MS approach.Although it is clear that LC-MS/MS provides a means of checking for the existence of specific components within a library, this approach begins to break down as a global strategy for characterizing complicated libraries. One problem that emerges involves the initial MS selection. Libraries are complex and can have many components that elute from the LC column at the same time. Thus, while specific ions from some components are being selected for fragmentation analysis other components eluting at the same time are often discarded (remaining unanalyzed). Additionally, combinatorial libraries present a challenge for MS because of mass-to-charge ratio (m/z) degeneracies that arise for isomeric components [4]. Consider for example, a simple mixture of three four-residue peptide sequence isomers (Glu-Val-Val-Leu, Val-Glu-Val-Leu, and ValVal-Glu-Leu). One might expect that initial m/z selection of these three isomers could be resolved based on fragmentation data-especially since accurate fragmentation pathways for peptides can often be predicted [5]. Table 1 lists the anticipated b-and y-type ions that are expected upon fragmentation of these three isomers. In this system, the existence of the Glu-Val-Val-Leu (EVVL) peptide can be unambiguously established based on the observation of peaks at m/z ϭ 130.0 and 330.2, which correspond to the E (b 1 ) and VVL (y 3 ) fragments; the presence of Val-Val-Glu-Leu (VVEL) can be established if peaks at m/z ϭ 199.1 and 261.1,

Section: Evidence For a Synthetic Failuresupporting

confidence: 61%

“…We have shown that it is possible to separate many different isomer types, including sequence-, structural-and stereo-isomers. 6 A key aspect of the separation is simply that different isomers establish different conformations (having different collision cross sections) in the gas phase.…”

mentioning

confidence: 99%

Development of LC-IMS-CID-TOFMS techniques: Analysis of a 256 component tetrapeptide combinatorial library

Hilderbrand

Myung

Barnes

et al. 2003

Self Cite

“…Ion mobility has been used extensively to separate ions of small organic molecules in the gas phase on the basis of collision cross section-to-charge ratios (⍀/z) [11][12][13][14][15][16]. IM/MS has been used to determine the conformations of mass identified peptide and protein ions as well to investigate the gas-phase dynamics of biological molecules [17][18][19][20]. Recently, ESI [14,15] and MALDI [10] have been used in combination with IM drift cells.…”

mentioning

confidence: 99%

A study of peptide—Peptide interactions using MALDI ion mobility o-TOF and ESI mass spectrometry

Woods

Koomen

Ruotolo

et al. 2002

Matrix-assisted laser desorption ionization ion mobility coupled to orthogonal time-of-flight mass spectrometry (MALDI-IM-oTOF MS) is evaluated as a tool for studying non-covalent complex (NCX) formation between peptides. The NCX formed between dynorphin 1-7 and Mini Gastrin I is used as a model system for comparison to previous MALDI experiments (Woods, A. S.; Huestis, M. A. J. Am. Soc. Mass Spectrom. 2001, 12, 88 -96). The dynorphin 1-7/Mini Gastrin I complex is stable after more than a ms drift time through the He filled mobility cell. Furthermore, the effects of solution pH on NCX ion signal intensity is measured both by MALDI-IM-MS analysis and by nanoelectrospray mass spectrometry. When compared to the previous MALDI study this work shows that all three techniques give similar results. In addition, fragmentation can be observed from of the non-covalent complex parent ion that occurs prior to TOF mass analysis but after mobility separation, thus providing NCX composition information. N on-covalent interactions have been studied previously, most often using electrospray ionization (ESI) mass spectrometry [1]. One of the motivations of this approach is the possibility that the structure and conformations of the gas-phase ions could retain clues to biological activity. However, non-covalent complexes are often in the presence of interferants (i.e., salts, detergents, or other solubilizing agents), which suppress ESI, making MALDI a more suitable ionization method. Many types of non-covalent complexes have also been successfully analyzed with MALDI as shown in Farmer and Caprioli's excellent review [2]. Furthermore, developing the ability to directly identify non-covalent complexes on a bio-surface such as a tissue slice or a protein array depends on first demonstrating the capability of MALDI analysis of complexes by testing it on model systems.A recent study of peptide-peptide interactions [3] has demonstrated that MALDI can be used to observe the formation of non-covalent complexes involving a salt bridge between an acidic peptide containing two or more adjacent Glu or Asp and a basic peptide containing two or more adjacent Arg or the Arg-Lys-Arg motif. The guanido group of the Arg side chain has a net positive charge that can be neutralized by removing a proton while the carboxyl groups of Asp and Glu have resonance structures where one pair of electrons from each oxygen is delocalized over the molecular orbital system. Therefore, the proton from the Arg side chain is attracted to the delocalized lone pair of electrons on the Asp or Glu side chain carboxyl group [4]. These interactions between very close oppositely charged groups in peptides and proteins are known as salt bridges [5].The detection of such complexes by MALDI is strongly dependent on the peptide-matrix solution pH [3, 6 -8]. Anfinsen [9] has shown that loss of biological function can come about through disruption of the tertiary structure resulting from pH, temperature, or pressure, leading to the disruption of non-covalent bonds. In this wo...

“…This is interesting because the trans-addition of the PEG arm does not significantly alter the shape of the europium complex. The 2-and 1-arm Eu-PEG products having closely related shapes provides an explanation for difficulties in purification using SEC, highlighting the utility of using three-dimensional separation by charge, size, and shape inherent to IMS [54,72].…”

Section: Ims Separationmentioning

confidence: 98%

Matrix-Assisted Ionization-Ion Mobility Spectrometry-Mass Spectrometry: Selective Analysis of a Europium–PEG Complex in a Crude Mixture

Fischer

Lutomski

El‐Baba

et al. 2015

Abstract. The analytical utility of a new and simple to use ionization method, matrixassisted ionization (MAI), coupled with ion mobility spectrometry (IMS) and mass spectrometry (MS) is used to characterize a 2-armed europium(III)-containing poly(-ethylene glycol) (Eu-PEG) complex directly from a crude sample. MAI was used with the matrix 1,2-dicyanobenzene, which affords low chemical background relative to matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). MAI provides high ion abundance of desired products in comparison to ESI and MALDI. Inductively coupled plasma-MS measurements were used to estimate a maximum of 10% of the crude sample by mass was the 2-arm Eu-PEG complex, supporting evidence of selective ionization of Eu-PEG complexes using the new MAI matrix, 1,2-dicyanobenzene. Multiply charged ions formed in MAI enhance the IMS gas-phase separation, especially relative to the singly charged ions observed with MALDI. Individual components are cleanly separated and readily identified, allowing characterization of the 2-arm Eu-PEG conjugate from a mixture of the 1-arm Eu-PEG complex and unreacted starting materials. Size-exclusion chromatography, liquid chromatography at critical conditions, MALDI-MS, ESI-MS, and ESI-IMS-MS had difficulties with this analysis, or failed.