Lloyd M. Smith

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Name: Smith, Lloyd M.

Organization: University of Wisconsin–Madison , USA

Department: Department of Chemistry

Title: Professor(PhD)

TOPICS

Analytical Chemistry

Chemicals
References

Global Identification of Protein Post-translational Modifications in a Single-Pass Database Search

Co-reporter:Michael R. Shortreed;Craig D. Wenger;Brian L. Frey;Mark Scalf;Gloria M. Sheynkman;Mark P. Keller;Alan D. Attie

Journal of Proteome Research November 6, 2015 Volume 14(Issue 11) pp:4714-4720

Publication Date(Web):Publication Date (Web): September 7, 2015

DOI:10.1021/acs.jproteome.5b00599

Bottom-up proteomics database search algorithms used for peptide identification cannot comprehensively identify post-translational modifications (PTMs) in a single-pass because of high false discovery rates (FDRs). A new approach to database searching enables global PTM (G-PTM) identification by exclusively looking for curated PTMs, thereby avoiding the FDR penalty experienced during conventional variable modification searches. We identified over 2200 unique, high-confidence modified peptides comprising 26 different PTM types in a single-pass database search.Keywords: acetylation; database search; G-PTM; Jurkat; Morpheus; phosphorylation; post-translational modification; proteomics; PTM;

Elucidating Escherichia coli Proteoform Families Using Intact-Mass Proteomics and a Global PTM Discovery Database

Co-reporter:Yunxiang Dai, Michael R. Shortreed, Mark Scalf, Brian L. Frey, Anthony J. Cesnik, Stefan Solntsev, Leah V. Schaffer, and Lloyd M. Smith

Journal of Proteome Research November 3, 2017 Volume 16(Issue 11) pp:4156-4156

Publication Date(Web):October 2, 2017

DOI:10.1021/acs.jproteome.7b00516

A proteoform family is a group of related molecular forms of a protein (proteoforms) derived from the same gene. We have previously described a strategy to identify proteoforms and elucidate proteoform families in complex mixtures of intact proteins. The strategy is based upon measurements of two properties for each proteoform: (i) the accurate proteoform intact-mass, measured by liquid chromatography/mass spectrometry (LC–MS), and (ii) the number of lysine residues in each proteoform, determined using an isotopic labeling approach. These measured properties are then compared with those extracted from a catalog of theoretical proteoforms containing protein sequences and localized post-translational modifications (PTMs) for the organism under study. A match between the measured properties and those in the catalog constitutes an identification of the proteoform. In the present study, this strategy is extended by utilizing a global PTM discovery database and is applied to the widely studied model organism Escherichia coli, providing the most comprehensive elucidation of E. coli proteoforms and proteoform families to date.Keywords: database search; E. coli; intact-mass; NeuCode; proteoform; proteoform family; PTM;

Multiplexed Sequence-Specific Capture of Chromatin and Mass Spectrometric Discovery of Associated Proteins

Co-reporter:Yunxiang Dai, Julia Kennedy-Darling, Michael R. Shortreed, Mark Scalf, Audrey P. Gasch, and Lloyd M. Smith

Analytical Chemistry August 1, 2017 Volume 89(Issue 15) pp:7841-7841

Publication Date(Web):June 27, 2017

DOI:10.1021/acs.analchem.7b01784

Comprehensive understanding of a gene’s expression and regulation at the molecular level requires identification of all proteins interacting with the gene. HyCCAPP (Hybridization Capture of Chromatin Associated Proteins for Proteomics) is an approach that uses single-stranded DNA oligonucleotides to capture specific genomic sequences in cross-linked chromatin fragments and identify associated proteins by mass spectrometry. Previous studies have shown HyCCAPP to provide useful information on protein–DNA interactions, revealing the proteins associated with the GAL1-10 region in yeast. We present here a multiplexed version of HyCCAPP. Utilizing a toehold-mediated capture/release strategy, HyCCAPP is targeted to multiple genomic loci in parallel, and the protein binders at each locus are eluted in a programmable and selective fashion. Multiplexed HyCCAPP was applied to four genes (25S rDNA, ARX1, CTT1, and RPL30) in S. cerevisiae under normal and stressed conditions. Capture and release efficiencies and specificities were comparable to those obtained without multiplexing. Using mass spectrometry-based bottom-up proteomics, hundreds of proteins were discovered at each locus in each condition. Statistical analysis revealed 34–88 enriched proteins in each gene capture. Many of these proteins had expected functions, including DNA-related and ribosome biogenesis-associated activities. Multiplexed HyCCAPP provides a useful strategy for the identification of proteins interacting with specific chromatin regions.

Discovery of Chromatin-Associated Proteins via Sequence-Specific Capture and Mass Spectrometric Protein Identification in Saccharomyces cerevisiae

Co-reporter:Julia Kennedy-Darling;Hector Guillen-Ahlers;Michael R. Shortreed;Mark Scalf;Brian L. Frey;Christina Kendziorski;Michael Olivier;Audrey P. Gasch

Journal of Proteome Research August 1, 2014 Volume 13(Issue 8) pp:3810-3825

Publication Date(Web):2017-2-22

DOI:10.1021/pr5004938

DNA–protein interactions play critical roles in the control of genome expression and other fundamental processes. An essential element in understanding how these systems function is to identify their molecular components. We present here a novel strategy, Hybridization Capture of Chromatin Associated Proteins for Proteomics (HyCCAPP), to identify proteins that are interacting with any given region of the genome. This technology identifies and quantifies the proteins that are specifically interacting with a genomic region of interest by sequence-specific hybridization capture of the target region from in vivo cross-linked chromatin, followed by mass spectrometric identification and quantification of associated proteins. We demonstrate the utility of HyCCAPP by identifying proteins associated with three multicopy and one single-copy loci in yeast. In each case, a locus-specific pattern of target-associated proteins was revealed. The binding of previously unknown proteins was confirmed by ChIP in 11 of 17 cases. The identification of many previously known proteins at each locus provides strong support for the ability of HyCCAPP to correctly identify DNA-associated proteins in a sequence-specific manner, while the discovery of previously unknown proteins provides new biological insights into transcriptional and regulatory processes at the target locus.Keywords: ChIP; chromatin; chromatin immunoprecipitation; DNA-binding proteins; DNA−protein interactions; GENECAPP; hybridization; HyCCAPP; mass spectrometry; proteomics; rDNA; ribosome biogenesis; transcription factors; transcriptional regulation; X-element;

Global Post-Translational Modification Discovery

Co-reporter:Qiyao Li, Michael R. Shortreed, Craig D. Wenger, Brian L. Frey, Leah V. Schaffer, Mark Scalf, and Lloyd M. Smith

Journal of Proteome Research April 7, 2017 Volume 16(Issue 4) pp:1383-1383

Publication Date(Web):November 22, 2016

DOI:10.1021/acs.jproteome.6b00034

A new global post-translational modification (PTM) discovery strategy, G-PTM-D, is described. A proteomics database containing UniProt-curated PTM information is supplemented with potential new modification types and sites discovered from a first-round search of mass spectrometry data with ultrawide precursor mass tolerance. A second-round search employing the supplemented database conducted with standard narrow mass tolerances yields deep coverage and a rich variety of peptide modifications with high confidence in complex unenriched samples. The G-PTM-D strategy represents a major advance to the previously reported G-PTM strategy and provides a powerful new capability to the proteomics research community.Keywords: database search; G-PTM-D; post-translational modification discovery; proteomics;

Human Proteomic Variation Revealed by Combining RNA-Seq Proteogenomics and Global Post-Translational Modification (G-PTM) Search Strategy

Co-reporter:Anthony J. Cesnik; Michael R. Shortreed; Gloria M. Sheynkman; Brian L. Frey

Journal of Proteome Research 2016 Volume 15(Issue 3) pp:800-808

Publication Date(Web):December 25, 2015

DOI:10.1021/acs.jproteome.5b00817

Mass-spectrometry-based proteomic analysis underestimates proteomic variation due to the absence of variant peptides and posttranslational modifications (PTMs) from standard protein databases. Each individual carries thousands of missense mutations that lead to single amino acid variants, but these are missed because they are absent from generic proteomic search databases. Myriad types of protein PTMs play essential roles in biological processes but remain undetected because of increased false discovery rates in variable modification searches. We address these two fundamental shortcomings of bottom-up proteomics with two recently developed software tools. The first consists of workflows in Galaxy that mine RNA sequencing data to generate sample-specific databases containing variant peptides and products of alternative splicing events. The second tool applies a new strategy that alters the variable modification approach to consider only curated PTMs at specific positions, thereby avoiding the combinatorial explosion that traditionally leads to high false discovery rates. Using RNA-sequencing-derived databases with this Global Post-Translational Modification (G-PTM) search strategy revealed hundreds of single amino acid variant peptides, tens of novel splice junction peptides, and several hundred posttranslationally modified peptides in each of ten human cell lines.

Elucidating Proteoform Families from Proteoform Intact-Mass and Lysine-Count Measurements

Co-reporter:Michael R. Shortreed; Brian L. Frey; Mark Scalf; Rachel A. Knoener; Anthony J. Cesnik

Journal of Proteome Research 2016 Volume 15(Issue 4) pp:1213-1221

Publication Date(Web):March 4, 2016

DOI:10.1021/acs.jproteome.5b01090

Proteomics is presently dominated by the “bottom-up” strategy, in which proteins are enzymatically digested into peptides for mass spectrometric identification. Although this approach is highly effective at identifying large numbers of proteins present in complex samples, the digestion into peptides renders it impossible to identify the proteoforms from which they were derived. We present here a powerful new strategy for the identification of proteoforms and the elucidation of proteoform families (groups of related proteoforms) from the experimental determination of the accurate proteoform mass and number of lysine residues contained. Accurate proteoform masses are determined by standard LC–MS analysis of undigested protein mixtures in an Orbitrap mass spectrometer, and the lysine count is determined using the NeuCode isotopic tagging method. We demonstrate the approach in analysis of the yeast proteome, revealing 8637 unique proteoforms and 1178 proteoform families. The elucidation of proteoforms and proteoform families afforded here provides an unprecedented new perspective upon proteome complexity and dynamics.

Photolithographic Synthesis of High-Density DNA and RNA Arrays on Flexible, Transparent, and Easily Subdivided Plastic Substrates

Co-reporter:Matthew T. Holden, Matthew C. D. Carter, Cheng-Hsien Wu, Jamison Wolfer, Eric Codner, Michael R. Sussman, David M. Lynn, and Lloyd M. Smith

Analytical Chemistry 2015 Volume 87(Issue 22) pp:11420

Publication Date(Web):October 23, 2015

DOI:10.1021/acs.analchem.5b02893

The photolithographic fabrication of high-density DNA and RNA arrays on flexible and transparent plastic substrates is reported. The substrates are thin sheets of poly(ethylene terephthalate) (PET) coated with cross-linked polymer multilayers that present hydroxyl groups suitable for conventional phosphoramidite-based nucleic acid synthesis. We demonstrate that by modifying array synthesis procedures to accommodate the physical and chemical properties of these materials, it is possible to synthesize plastic-backed oligonucleotide arrays with feature sizes as small as 14 μm × 14 μm and feature densities in excess of 125 000/cm2, similar to specifications attainable using rigid substrates such as glass or glassy carbon. These plastic-backed arrays are tolerant to a wide range of hybridization temperatures, and improved synthetic procedures are described that enable the fabrication of arrays with sequences up to 50 nucleotides in length. These arrays hybridize with S/N ratios comparable to those fabricated on otherwise identical arrays prepared on glass or glassy carbon. This platform supports the enzymatic synthesis of RNA arrays and proof-of-concept experiments are presented showing that the arrays can be readily subdivided into smaller arrays (or “millichips”) using common laboratory-scale laser cutting tools. These results expand the utility of oligonucleotide arrays fabricated on plastic substrates and open the door to new applications for these important bioanalytical tools.

Measuring the Formaldehyde Protein–DNA Cross-Link Reversal Rate

Co-reporter:Julia Kennedy-Darling and Lloyd M. Smith

Analytical Chemistry 2014 Volume 86(Issue 12) pp:5678

Publication Date(Web):May 21, 2014

DOI:10.1021/ac501354y

Protein–DNA binding interactions play critical roles in important cellular processes such as gene expression, cell division, and chromosomal organization. Techniques to identify and characterize these interactions often utilize formaldehyde cross-linking for stabilization of the complexes. Advantages of formaldehyde as a cross-linking reagent include cell permeability, relatively fast cross-linking kinetics, and short cross-linker length. In addition, formaldehyde cross-links are reversible, which has the advantage of allowing complexes to be dissociated if desired but may also present a problem if undesired dissociation occurs in the course of an experiment. While the kinetics of formaldehyde cross-link formation have been well-established in numerous studies, there have been no reports of the rate of cross-link dissociation, even though it is clearly a critical variable when developing a biochemical protocol involving formaldehyde cross-linking. We present here a method for measurement of the rate of formaldehyde cross-link reversal based upon the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) procedure and use it to determine the rate of cross-link reversal for cross-linked protein–DNA complexes from yeast cell lysate. The half-life of the protein–DNA cross-links varies from 179 h at 4 °C to 11.3 h at 47 °C, with a rate that increases exponentially with temperature and is independent of salt concentration.

Large-Scale Mass Spectrometric Detection of Variant Peptides Resulting from Nonsynonymous Nucleotide Differences

Co-reporter:Gloria M. Sheynkman, Michael R. Shortreed, Brian L. Frey, Mark Scalf, and Lloyd M. Smith

Journal of Proteome Research 2014 Volume 13(Issue 1) pp:228-240

Publication Date(Web):2017-2-22

DOI:10.1021/pr4009207

Each individual carries thousands of nonsynonymous single nucleotide variants (nsSNVs) in their genome, each corresponding to a single amino acid polymorphism (SAP) in the encoded proteins. It is important to be able to directly detect and quantify these variations at the protein level to study post-transcriptional regulation, differential allelic expression, and other important biological processes. However, such variant peptides are not generally detected in standard proteomic analyses due to their absence from the generic databases that are employed for mass spectrometry searching. Here we extend previous work that demonstrated the use of customized SAP databases constructed from sample-matched RNA-Seq data. We collected deep-coverage RNA-Seq data from the Jurkat cell line, compiled the set of nsSNVs that are expressed, used this information to construct a customized SAP database, and searched it against deep-coverage shotgun MS data obtained from the same sample. This approach enabled the detection of 421 SAP peptides mapping to 395 nsSNVs. We compared these peptides to peptides identified from a large generic search database containing all known nsSNVs (dbSNP) and found that more than 70% of the SAP peptides from this dbSNP-derived search were not supported by the RNA-Seq data and thus are likely false positives. Next, we increased the SAP coverage from the RNA-Seq derived database by utilizing multiple protease digestions, thereby increasing variant detection to 695 SAP peptides mapping to 504 nsSNV sites. These detected SAP peptides corresponded to moderate to high abundance transcripts (30+ transcripts per million, TPM). The SAP peptides included 192 allelic pairs; the relative expression levels of the two alleles were evaluated for 51 of those pairs and were found to be comparable in all cases.

Multiplexed Programmable Release of Captured DNA

Co-reporter:Julia Kennedy-Darling;Matthew T. Holden;Dr. Michael R. Shortreed; Lloyd M. Smith

ChemBioChem 2014 Volume 15( Issue 16) pp:2353-2356

Publication Date(Web):

DOI:10.1002/cbic.201402343

Abstract

Nucleic-acid hybridization is widely used for the specific capture of complementary sequences from complex samples. It is useful for both analytical methodologies, such as array hybridization (e.g. transcriptome analysis, genetic-variation analysis), and preparative strategies such as exome sequencing and sequence-specific proteome capture and analysis (PICh, HyCCAPP). It has not generally been possible to selectively elute particular captured subsequences, however, as the conditions employed for disruption of a duplex can lack the specificity needed to discriminate between different sequences. We show here that it is possible to bind and selectively release multiple sets of sequences by using toehold-mediated DNA branch migration. The strategy is illustrated for simple mixtures of oligonucleotides, for the sequence-specific capture and specific release of crosslinked yeast chromatin, and for the specific release of oligonucleotides hybridized to DNA microarrays.

Inside Cover: Multiplexed Programmable Release of Captured DNA (ChemBioChem 16/2014)

Co-reporter:Julia Kennedy-Darling;Matthew T. Holden;Dr. Michael R. Shortreed; Lloyd M. Smith

ChemBioChem 2014 Volume 15( Issue 16) pp:

Publication Date(Web):

DOI:10.1002/cbic.201490056

Enzymatic Fabrication of High-Density RNA Arrays

Co-reporter:Dr. Cheng-Hsien Wu;Matthew T. Holden; Lloyd M. Smith

Angewandte Chemie 2014 Volume 126( Issue 49) pp:13732-13735

Publication Date(Web):

DOI:10.1002/ange.201408747

Abstract

A powerful new strategy for the fabrication of high-density RNA arrays is described. A high-density DNA array is fabricated by standard photolithographic methods, the surface-bound DNA molecules are enzymatically copied into their RNA complements from a surface-bound RNA primer, and the DNA templates are enzymatically destroyed, leaving behind the desired RNA array. The strategy is compatible with 2′-fluoro-modified (2′F) ribonucleoside triphosphates (rNTPs), which may be included in the polymerase extension reaction to impart nuclease resistance and other desirable characteristics to the synthesized RNAs. The use and fidelity of the arrays are explored with DNA hybridization, DNAzyme cleavage, and nuclease digestion experiments.

Detection of Large Ions in Time-of-Flight Mass Spectrometry: Effects of Ion Mass and Acceleration Voltage on Microchannel Plate Detector Response

Co-reporter:Ranran Liu;Qiyao Li

Journal of The American Society for Mass Spectrometry 2014 Volume 25( Issue 8) pp:1374-1383

Publication Date(Web):2014 August

DOI:10.1007/s13361-014-0903-2

In time-of-flight mass spectrometry (TOF-MS), ion detection is typically accomplished by the generation and amplification of secondary electrons produced by ions colliding with a microchannel plate (MCP) detector. Here, the response of an MCP detector as a function of ion mass and acceleration voltage is characterized, for singly charged peptide/protein ions ranging from 1 to 290 kDa in mass, and for acceleration voltages from 5 to 25 kV. A nondestructive inductive charge detector (ICD) employed in parallel with MCP detection provides a reliable reference signal to allow accurate calibration of the MCP response. MCP detection efficiencies were very close to unity for smaller ions at high acceleration voltages (e.g., angiotensin, 1046.5 Da, at 25 kV acceleration voltage), but decreased to ~11% for the largest ions examined (immunoglobulin G (IgG) dimer, 290 kDa) even at the highest acceleration voltage employed (25 kV). The secondary electron yield γ (average number of electrons produced per ion collision) is found to be proportional to mv3.1 (m: ion mass, v: ion velocity) over the entire mass range examined, and inversely proportional to the square root of m in TOF-MS analysis. The results indicate that although MCP detectors indeed offer superlative performance in the detection of smaller peptide/protein species, their performance does fall off substantially for larger proteins, particularly under conditions of low acceleration voltage.

Enzymatic Fabrication of High-Density RNA Arrays

Co-reporter:Dr. Cheng-Hsien Wu;Matthew T. Holden; Lloyd M. Smith

Angewandte Chemie International Edition 2014 Volume 53( Issue 49) pp:13514-13517

Publication Date(Web):

DOI:10.1002/anie.201408747

Abstract

Chemical Derivatization of Peptide Carboxyl Groups for Highly Efficient Electron Transfer Dissociation

Co-reporter:Brian L. Frey;Daniel T. Ladror

Journal of The American Society for Mass Spectrometry 2013 Volume 24( Issue 11) pp:1710-1721

Publication Date(Web):2013 November

DOI:10.1007/s13361-013-0701-2

The carboxyl groups of tryptic peptides were derivatized with a tertiary or quaternary amine labeling reagent to generate more highly charged peptide ions that fragment efficiently by electron transfer dissociation (ETD). All peptide carboxyl groups—aspartic and glutamic acid side-chains as well as C-termini—were derivatized with an average reaction efficiency of 99 %. This nearly complete labeling avoids making complex peptide mixtures even more complex because of partially-labeled products, and it allows the use of static modifications during database searching. Alkyl tertiary amines were found to be the optimal labeling reagent among the four types tested. Charge states are substantially higher for derivatized peptides: a modified tryptic digest of bovine serum albumin (BSA) generates ~90% of its precursor ions with z > 2, compared with less than 40 % for the unmodified sample. The increased charge density of modified peptide ions yields highly efficient ETD fragmentation, leading to many additional peptide identifications and higher sequence coverage (e.g., 70 % for modified versus only 43 % for unmodified BSA). The utility of this labeling strategy was demonstrated on a tryptic digest of ribosomal proteins isolated from yeast cells. Peptide derivatization of this sample produced an increase in the number of identified proteins, a >50 % increase in the sequence coverage of these proteins, and a doubling of the number of peptide spectral matches. This carboxyl derivatization strategy greatly improves proteome coverage obtained from ETD-MS/MS of tryptic digests, and we anticipate that it will also enhance identification and localization of post-translational modifications.

In situ Synthesis of Oligonucleotide Arrays on Surfaces Coated with Crosslinked Polymer Multilayers

Co-reporter:Adam H. Broderick, Matthew R. Lockett, Maren E. Buck, Yuan Yuan, Lloyd M. Smith, and David M. Lynn

Chemistry of Materials 2012 Volume 24(Issue 5) pp:938

Publication Date(Web):November 28, 2011

DOI:10.1021/cm202720q

We report an approach to the in situ synthesis of oligonucleotide arrays on surfaces coated with crosslinked polymer multilayers. Our approach makes use of methods for the “reactive” layer-by-layer assembly of thin, amine-reactive multilayers using branched polyethyleneimine (PEI) and the azlactone-functionalized polymer poly(2-vinyl-4,4′-dimethylazlactone) (PVDMA). Postfabrication treatment of film-coated glass substrates with d-glucamine or 4-amino-1-butanol yielded hydroxyl-functionalized films suitable for the Maskless Array Synthesis (MAS) of oligonucleotide arrays. Glucamine-functionalized films yielded arrays of oligonucleotides with fluorescence intensities and signal-to-noise ratios (after hybridization with fluorescently labeled complementary strands) comparable to those of arrays fabricated on conventional silanized glass substrates. These arrays could be exposed to multiple hybridization/dehybridization cycles with only moderate loss of hybridization density. The versatility of the layer-by-layer approach also permitted synthesis directly on thin sheets of film-coated poly(ethylene terephthalate) (PET) to yield flexible oligonucleotide arrays that could be readily manipulated (e.g., bent) and cut into smaller arrays. To our knowledge, this work presents the first use of polymer multilayers as a substrate for the multistep synthesis of complex molecules. Our results demonstrate that these films are robust and able to withstand the ∼450 individual chemical processing steps associated with MAS (as well as manipulations required to hybridize, image, and dehybridize the arrays) without large-scale cracking, peeling, or delamination of the thin films. The combination of layer-by-layer assembly and MAS provides a means of fabricating functional oligonucleotide arrays on a range of different materials and substrates. This approach may also prove useful for the fabrication of supports for the solid-phase synthesis and screening of other macromolecular or small-molecule agents.Keywords: in situ synthesis; layer-by-layer; Maskless Array Synthesis; oligonucleotide arrays; polymer multilayers; reactive assembly; thin films;

RNA-Mediated Gene Assembly from DNA Arrays

Co-reporter:Dr. Cheng-Hsien Wu;Dr. Matthew R. Lockett ; Lloyd M. Smith

Angewandte Chemie 2012 Volume 124( Issue 19) pp:4706-4710

Publication Date(Web):

DOI:10.1002/ange.201109058

RNA-Mediated Gene Assembly from DNA Arrays

Co-reporter:Dr. Cheng-Hsien Wu;Dr. Matthew R. Lockett ; Lloyd M. Smith

Angewandte Chemie International Edition 2012 Volume 51( Issue 19) pp:4628-4632

Publication Date(Web):

DOI:10.1002/anie.201109058

Parallel Detection of Intrinsic Fluorescence from Peptides and Proteins for Quantification during Mass Spectrometric Analysis

Co-reporter:Jason D. Russell, Ryan T. Hilger, Daniel T. Ladror, Mark A. Tervo, Mark Scalf, Michael R. Shortreed, Joshua J. Coon, and Lloyd M. Smith

Analytical Chemistry 2011 Volume 83(Issue 6) pp:2187

Publication Date(Web):February 11, 2011

DOI:10.1021/ac103023q

Direct mass spectrometric quantification of peptides and proteins is compromised by the wide variabilities in ionization efficiency which are hallmarks of both the MALDI and ESI ionization techniques. We describe here the implementation of a fluorescence detection system for measurement of the UV-excited intrinsic fluorescence (UV-IF) from peptides and proteins just prior to their exit and electrospray ionization from an ESI capillary. The fluorescence signal provides a quantifiable measure of the amount of protein or peptide present, while direct or tandem mass spectrometric analysis (MS/MS) on the ESI-generated ions provides information on identity. We fabricated an inexpensive, modular fluorescence excitation and detection device utilizing an ultraviolet light-emitting diode for excitation in a ∼300 nL fluorescence detection cell integrated into the fused-silica separation column. The fluorescence signal is linear over 3 orders of magnitude with on-column limits of detection in the low femtomole range. Chromatographically separated intact proteins analyzed using UV-IF prior to top-down mass spectrometry demonstrated sensitive detection of proteins as large as 77 kDa.

To understand the whole, you must know the parts: unraveling the roles of protein–DNA interactions in genome regulation

Co-reporter:Lloyd M. Smith, Michael R. Shortreed and Michael Olivier

Analyst 2011 vol. 136(Issue 15) pp:3060-3065

Publication Date(Web):01 Jun 2011

DOI:10.1039/C1AN15037E

The regulation of gene transcription is fundamental to the existence of complex multicellular organisms such as humans. This process dictates which genes are expressed in which tissues, and controls how various cell types grow, differentiate, and respond to their environments. Although the deciphering of the human genome sequence has given us the “source code” for life, we still know far too little about the mechanisms that control which sets of genes are active in which tissues, and how their expression is regulated. It is clear, however, that much of this system depends upon the sequence-specific interactions of regulatory proteins with particular genetic loci. To be able to unravel the details of these interactions on a genome-wide basis, it is necessary to know what proteins are bound to the DNA where in the genome, and to be able to monitor how those proteins change over time and in response to external stimuli. Developing a new technology to provide this information constitutes a “Grand Challenge” for Analytical Chemistry. In this brief article we outline the nature of this challenge, and propose one strategy to address it.

Alkylating Tryptic Peptides to Enhance Electrospray Ionization Mass Spectrometry Analysis

Co-reporter:Suzanne E. Kulevich, Brian L. Frey, Gloria Kreitinger, and Lloyd M. Smith

Analytical Chemistry 2010 Volume 82(Issue 24) pp:10135

Publication Date(Web):November 29, 2010

DOI:10.1021/ac1019792

A major limitation of mass spectrometry-based proteomics is inefficient and differential ionization during electrospray ionization (ESI). This leads to problems such as increased limits of detection and incomplete sequence coverage of proteins. Incomplete sequence coverage is especially problematic for analyses that require the detection and identification of specific peptides from a protein, such as the analysis of post-translational modifications. We describe here the development and use of aldehyde-based chemistry for the alkylation of peptide primary amines to increase peptide hydrophobicity, providing increased ionization efficiency and concomitant signal enhancement. When employed to modify the peptide products of protein tryptic digests, increased sequence coverage is obtained from combined modified and unmodified digests. To evaluate the utility of alkylation of peptides for selected reaction monitoring (SRM) assays, we alkylated a peptide from the protein Oct4, known to play a role in regulating stem cell differentiation. Increased chromatographic retention and ionization efficiency is observed for the alkylated Oct4 peptide compared to its unmodified form.

Halogenation of Carbon Substrates for Increased Reactivity with Alkenes

Co-reporter:Matthew R. Lockett and Lloyd M. Smith

Langmuir 2010 Volume 26(Issue 22) pp:16642-16646

Publication Date(Web):October 6, 2010

DOI:10.1021/la103050z

Carbon substrates are readily functionalized with alkene-containing molecules via an ultraviolet-light-catalyzed reaction, resulting in the formation of a carbon−carbon bond with the surface. This reaction is typically performed on hydrogen-terminated carbon substrates, limiting its utility as alkene molecules with low electron affinities do not readily attach to this surface. Recently, a wet-chemical method for preparing bromine- and chlorine-terminated carbon substrates has been developed. Replacing the terminal hydrogen atoms with a halogen analog increases the surface’s reactivity with alkene-containing molecules, affording a means of modifying the carbon substrate with the alkene molecules that do not readily attach to the hydrogen-terminated surface and with a greatly reduced reaction time.

Formation and Stability of Alkylthiol Monolayers on Carbon Substrates

Co-reporter:Matthew R. Lockett and Lloyd M. Smith

The Journal of Physical Chemistry C 2010 Volume 114(Issue 29) pp:12635-12641

Publication Date(Web):July 7, 2010

DOI:10.1021/jp102821x

The formation and stability of alkylthiol monolayers on amorphous carbon thin films are investigated. Alkylthiol monolayers were prepared via a two-step, wet chemical process in which the carbon surface was first halogenated and then incubated with (4-(trifluoromethyl)phenyl)methanethiol (4tBM). The 4tBM covalently attaches to the surface in a substitution reaction in which the 4tBM thiol replaces the surface halogen. Studies of the substitution mechanism showed that monolayer formation is affected by the nature of the surface-bound halogen as well as the concentration and nucleophilicity of the 4tBM sulfur atom, consistent with a bimolecular (SN2) substitution reaction mechanism. The alkylthiol monolayers are stable over a wide range of solvents, pH, and temperature conditions.

Rapid Determination of RNA Accessible Sites by Surface Plasmon Resonance Detection of Hybridization to DNA Arrays

Co-reporter:Joshua B. Mandir, Matthew R. Lockett, Margaret F. Phillips, Hatim T. Allawi, Victor I. Lyamichev and Lloyd M. Smith

Analytical Chemistry 2009 Volume 81(Issue 21) pp:8949

Publication Date(Web):October 7, 2009

DOI:10.1021/ac9015962

RNA accessible sites are the regions in an RNA molecule that are available for hybridization with cDNA or RNA molecules. The identification of these accessible sites is a critical first step in identifying antisense-mediated gene suppression sites, as well as in a variety of other RNA-based analysis methods. Here, we present a rapid, hybridization-based, label-free method of identifying RNA accessible sites with surface plasmon resonance imaging (SPRi) on in situ synthesized oligonucleotide arrays prepared on carbon-on-metal substrates. The accessible sites of three pre-miRNAs, miRNA precursors of ∼75 nt in length, were determined by hybridizing the RNA molecules to RNA-specific tiling arrays. An array composed of all possible 6mer oligonucleotide sequences was also utilized in this work, offering a universal platform capable of studying RNA molecules in a high throughput manner.

Single Droplet Separations and Surface Partition Coefficient Measurements Using Laser Ablation Mass Spectrometry

Co-reporter:Kaveh Jorabchi and Lloyd M. Smith

Analytical Chemistry 2009 Volume 81(Issue 23) pp:9682

Publication Date(Web):November 3, 2009

DOI:10.1021/ac901819r

Surface activity of analytes plays a significant role in many chemical and physical phenomena. We present here a mass spectrometric method to characterize surface activity and solute partitioning between bulk liquid and the gas−liquid interface in droplets. The approach employs ablation by an infrared (IR) laser from the surface of a microliter droplet deposited on a stainless steel post. The ablated material is ionized for mass spectrometric analysis by either droplet charging or by postionization in an electrospray plume. Three areas of application have been explored using this method (1) separations in a single droplet: continuous ablation by a series of many successive laser pulses results in faster depletion of more surface active analytes, effectively comprising a surface activity-based separation. (2) Partition coefficient measurements: droplet volume is held constant during ablation by continually replenishing lost solvent. This leads to analyte-specific ion signal decay curves that may be fitted to a model based on Langmuir adsorption isotherms and simple analytical expressions, yielding quantitative values for the analyte surface partition coefficients. (3) Studies of the relationship between surface partitioning and high-performance liquid chromatography (HPLC) phase partitioning: comparisons of surface activities measured by laser desorption with retention times in reversed-phase HPLC reveal that the relationship between the two partitioning processes is very sensitive to chemical structure. Poor correlation between the retention time and surface activity is also observed within a subcategory of analytes (peptides). This effect is attributed to multimodal solute−stationary phase interactions. The laser desorption approach presented here provides direct information on analyte surface activities free from the complications encountered in chromatographic methods due to chemical structure variations.

Acyl Chloride-Modified Amorphous Carbon Substrates for the Attachment of Alcohol-, Thiol-, and Amine-Containing Molecules

Co-reporter:Matthew R. Lockett, Justin C. Carlisle, Dinh V. Le and Lloyd M. Smith

Langmuir 2009 Volume 25(Issue 9) pp:5120-5126

Publication Date(Web):March 24, 2009

DOI:10.1021/la804140r

Amorphous carbon thin films are easily deposited at room temperature, readily functionalized with alkene-containing molecules through a UV photochemical reaction, and provide a robust surface capable of supporting array fabrication. Relatively few attachment chemistries for the fabrication of small organic molecule and/or biomolecule arrays on carbon substrates have been described to date. Here, acyl chloride-terminated amorphous carbon substrates were fabricated, characterized, and used to attach alcohol-, thiol-, and amine-containing small molecules. Oligonucleotide arrays of thiol- and amine-modified oligonucleotides were also prepared on these substrates. The hybridization density, average fluorescence signal of hybridized features, and average background fluorescence of oligonucleotide arrays prepared on acyl chloride-modified substrates were compared to the same parameters for oligonucleotide arrays prepared on maleimide- and aldehyde-modified substrates.

Controlling Oligonucleotide Surface Density in Light-Directed DNA Array Fabrication

Co-reporter:Siyuan Chen, Margaret F. Phillips, Franco Cerrina and Lloyd M. Smith

Langmuir 2009 Volume 25(Issue 11) pp:6570-6575

Publication Date(Web):March 12, 2009

DOI:10.1021/la9000297

Over the past two decades high-density DNA arrays have developed into a central technology for nucleic acid analyses. Important application areas include whole-genome gene expression studies, high throughput analyses of single nucleotide polymorphisms, and, most recently, the determination of binding site specificities for transcription factors and other critical elements involved in gene regulation. A key parameter in the performance of DNA arrays is the density of the surface-bound oligonucleotides, which strongly affects both thermodynamic and kinetic aspects of DNA hybridization. In this report, we describe an approach for the control of oligonucleotide density in photolithographically fabricated DNA arrays, based upon a controlled UV light deprotection procedure. Modulation of the UV exposure permits a desired degree of deprotection of surface synthesis sites; a subsequent capping reaction to inactivate the exposed sites leaves only a desired fraction of active sites remaining for synthesis, corresponding to a lower oligonucleotide density. It is shown that the procedure is reasonably general, in that it is readily transferable to alternative substrate materials with similar results.

Photopatterned Thiol Surfaces for Biomolecule Immobilization

Co-reporter:Siyuan Chen and Lloyd M. Smith

Langmuir 2009 Volume 25(Issue 20) pp:12275-12282

Publication Date(Web):September 4, 2009

DOI:10.1021/la9017135

The ability to pattern small molecules and proteins on artificial surfaces is of importance for the development of new tools including tissue engineering, cell-based drug screening, and cell-based sensors. We describe here a novel “caged” thiol-mediated strategy for the fabrication of planar substrates patterned with biomolecules using photolithography. A thiol-bearing phosphoramidite (3-(2′-nitrobenzyl)thiopropyl (NBTP) phosphoramidite) was synthesized and coupled to a hydroxyl-terminated amorphous carbon substrate. A biocompatible oligo(ethylene glycol) spacer was used to resist nonspecific adsorption of protein and DNA and enhance flexibility of attached biomolecules. Thiol functionalities are revealed by UV irradiation of NBTP-modified surfaces. Both the surface coupling and photodeprotection were monitored by Polarization Modulation Fourier Transform Infrared Reflection Absorption Spectroscopy (PM-FTIRRAS) and X-ray Photoelectron Spectroscopy (XPS) measurements. The newly exposed thiols are chemically very active and react readily with a wide variety of groups. A series of molecules including biotin, DNA, and proteins were attached to the surfaces with retention of their biological activities, demonstrating the utility and generality of the approach.

Attaching Molecules to Chlorinated and Brominated Amorphous Carbon Substrates via Grignard Reactions

Co-reporter:Matthew R. Lockett and Lloyd M. Smith

Langmuir 2009 Volume 25(Issue 6) pp:3340-3343

Publication Date(Web):February 11, 2009

DOI:10.1021/la8039626

Amorphous carbon thin films are an attractive material because they provide a chemically robust surface that has been utilized in biomolecule array, biosensor, and bioelectronic applications. These thin films are particularly versatile because they are deposited at room temperature, making them readily integrated with other materials and devices. Here we present an alternative means of functionalizing carbon substrates based on Grignard chemistry. First, the amorphous carbon substrates are halogenated with a solution-based, radical-initiated halogenation reaction using PX5 (X = Br or Cl). Next, the halogenated surfaces are modified via the formation of a carbon−carbon bond between the surface and the Grignard reagents employed. Alkyl-, perfluoroalkyl-, and poly(ethylene glycol)-Grignard reagents were chosen to show the utility of this reaction scheme in functionalizing carbon surfaces to withstand oxidation and provide a hydrophobic and/or biocompatible substrate.

Modifying the charge state distribution of proteins in electrospray ionization mass spectrometry by chemical derivatization

Co-reporter:Casey J. Krusemark;Brian L. Frey

Journal of The American Society for Mass Spectrometry 2009 Volume 20( Issue 9) pp:1617-1625

Publication Date(Web):2009 September

DOI:10.1016/j.jasms.2009.04.017

Electrospray ionization (ESI) of denatured proteins produces a broad distribution of multiply-charged ions leading to multiple peaks in the mass spectrum. We investigated changes in the positive-mode ESI charge state distribution produced by several chemical modifications of denatured proteins. Capping carboxylic acid groups with neutral functional groups yields little change in charge state distribution compared with unmodified proteins. The results indicate that carboxyl groups do not play a significant role in the positive charging of denatured proteins in ESI. The modification of proteins with additional basic sites or fixed positive charges generates substantially higher charge states, providing evidence that the number of ionizable sites, rather than molecular size and shape, determines ESI charging for denatured proteins. Fixed charge modification also significantly reduces the number of protons acquired by a protein, in that the charge state envelope is not increased by the full number of fixed charges appended. This result demonstrates that Coulombic repulsion between positive charges plays a significant role in determining charge state distribution by affecting the gas-phase basicity of ionizable sites. Addition of fixed-charge moieties to a protein is a useful approach for shifting protein charge state distributions to higher charge states, and with further work, it may help limit the distribution of protein ions to fewer charge states.

Mass Spectrometry of Acoustically Levitated Droplets

Co-reporter:Michael S. Westphall, Kaveh Jorabchi and Lloyd M. Smith

Analytical Chemistry 2008 Volume 80(Issue 15) pp:5847

Publication Date(Web):June 27, 2008

DOI:10.1021/ac800317f

Containerless sample handling techniques such as acoustic levitation offer potential advantages for mass spectrometry, by eliminating surfaces where undesired adsorption/desorption processes can occur. In addition, they provide a unique opportunity to study fundamental aspects of the ionization process as well as phenomena occurring at the air−droplet interface. Realizing these advantages is contingent, however, upon being able to effectively interface levitated droplets with a mass spectrometer, a challenging task that is addressed in this report. We have employed a newly developed charge and matrix-assisted laser desorption/ionization (CALDI) technique to obtain mass spectra from a 5-μL acoustically levitated droplet containing peptides and an ionic matrix. A four-ring electrostatic lens is used in conjunction with a corona needle to produce bursts of corona ions and to direct those ions toward the droplet, resulting in droplet charging. Analyte ions are produced from the droplet by a 337-nm laser pulse and detected by an atmospheric sampling mass spectrometer. The ion generation and extraction cycle is repeated at 20 Hz, the maximum operating frequency of the laser employed. It is shown in delayed ion extraction experiments that both positive and negative ions are produced, behavior similar to that observed for atmospheric pressure matrix-assisted laser absorption/ionization. No ion signal is observed in the absence of droplet charging. It is likely, although not yet proven, that the role of the droplet charging is to increase the strength of the electric field at the surface of the droplet, reducing charge recombination after ion desorption.

Ion–ion reactions with fixed-charge modified proteins to produce ions in a single, very high charge state

Co-reporter:Brian L. Frey, Casey J. Krusemark, Aaron R. Ledvina, Joshua J. Coon, Peter J. Belshaw, Lloyd M. Smith

International Journal of Mass Spectrometry 2008 Volume 276(2–3) pp:136-143

Publication Date(Web):1 October 2008

DOI:10.1016/j.ijms.2008.07.029

Electrospray ionization (ESI) of denatured proteins produces a mass spectrum with a broad distribution of multiply charged ions. Attaching fixed positive charges, specifically quaternary ammonium groups, to proteins at their carboxylic acid groups generates substantially higher charge states compared to the corresponding unmodified proteins in positive-mode ESI. Ion–ion reactions of these modified proteins with reagent anions leads to charge reduction by proton transfer. These proton transfer reactions cannot remove charge from the quaternary ammonium groups, which do not have a proton to transfer to the anion. Thus, one might expect charge reduction to stop at a single charge state equal to the number of fixed charges on the modified protein. However, ion–ion reactions yield charge states lower than this number of fixed charges due to anion attachment (adduction) to the proteins. Charge reduction via ion–molecule reactions involving gas-phase bases also give adducts on the modified protein ions in low charge states. Such adducts are avoided by keeping the ions in charge states well above the number of fixed charges. In the present work protein ions were selectively “parked” within an ion trap mass spectrometer in a high charge state by mild radiofrequency excitation that dramatically slows their ion–ion reaction rate—a technique termed “ion parking”. The combination of ion parking with the fixed-charge modified proteins permits generation of a large population of ions in a single, very high charge state.

Charge assisted laser desorption/ionization mass spectrometry of droplets

Co-reporter:Kaveh Jorabchi;Michael S. Westphall

Journal of The American Society for Mass Spectrometry 2008 Volume 19( Issue 6) pp:833-840

Publication Date(Web):2008 June

DOI:10.1016/j.jasms.2008.02.012

We propose and evaluate a new mechanism to account for analyte ion signal enhancement in ultraviolet-laser desorption mass spectrometry of droplets in the presence of corona ions. Our new insights are based on timing control of corona ion production, laser desorption, and peptide ion extraction achieved by a novel pulsed corona apparatus. We demonstrate that droplet charging rather than gas-phase ion-neutral reactions is the major contributor to analyte ion generation from an electrically isolated droplet. Implications of the new mechanism, termed charge assisted laser desorption/ionization (CALDI), are discussed and contrasted with those of the laser desorption atmospheric pressure chemical ionization method (LD-APCI). It is also demonstrated that analyte ion generation in CALDI occurs with external electric fields about one order of magnitude lower than those needed for atmospheric pressure matrix assisted laser desorption/ionization or electrospray ionization of droplets.

Is charge reduction in ESI really necessary?

Co-reporter:Lloyd M. Smith

Journal of The American Society for Mass Spectrometry 2008 Volume 19( Issue 5) pp:629-631

Publication Date(Web):2008 May

DOI:10.1016/j.jasms.2008.02.002

The technique of charge reduction electrospray mass spectrometry (CREMS), which can reduce the charge state complexity produced in electrospray ionization (ESI), is discussed.

Aldehyde-Terminated Amorphous Carbon Substrates for the Fabrication of Biomolecule Arrays

Co-reporter:Matthew R. Lockett, Michael R. Shortreed and Lloyd M. Smith

Langmuir 2008 Volume 24(Issue 17) pp:9198-9203

Publication Date(Web):August 2, 2008

DOI:10.1021/la800991t

Amorphous carbon thin films are easily deposited at room temperature, readily functionalized with alkene-containing molecules through a UV photochemical reaction, and provide a robust surface capable of supporting chemical and biomolecule array fabrication. Aldehyde-terminated amorphous carbon substrates were fabricated via the attachment of a 2-(10-undecen-1-yl)-1,3-dioxolane molecule. The surfaces were then deprotected in 1.5 M HCl to yield an aldehyde-terminated surface that is readily reactive with amine containing molecules. An array of amine-modified oligonucleotides was prepared on aldehyde-terminated surfaces prepared on both amorphous carbon and on gold self-assembled monolayers, and the fluorescence background, feature signal-to-noise ratio, and hybridization densities were compared. The aldehyde-terminated amorphous carbon substrates offer inherently lower background fluorescence intensity and a greater number of hybridization-accessible sites.

Characterization of vascular endothelial growth factor receptors on the endothelial cell surface during hypoxia using whole cell binding arrays

Co-reporter:Ji Eun Lee, Daniela N. Didier, Matthew R. Lockett, Mark Scalf, Andrew S. Greene, Michael Olivier, Lloyd M. Smith

Analytical Biochemistry 2007 Volume 369(Issue 2) pp:241-247

Publication Date(Web):15 October 2007

DOI:10.1016/j.ab.2007.06.010

Angiogenesis plays a central role in a variety of important biological processes such as reproduction, tissue development, and wound healing, as well as being critical to tumor formation in cancer. The development of chromosomal substitution (consomic) rat strains has permitted the chromosomal localization of genetic factors critical to angiogenesis, but many questions remain as to the mechanisms involved. Here we utilize a novel cell capture assay to assess changes in the functional expression of vascular endothelial growth factor (VEGF) receptors on the surface of vascular endothelial cells isolated from rat strains that are normal or impaired in angiogenesis. We show that functional VEGF receptor expression is increased under hypoxic conditions in rat strains that exhibit normal angiogenesis but not in a strain impaired in angiogenesis. This result implicates the dysregulation of VEGF receptor expression levels on the endothelial cell surface as a key factor in impaired angiogenesis.

Lectin-modified microchannels for mammalian cell capture and purification

Co-reporter:Ting Zheng;Hongmei Yu;Caroline M. Alexander;David J. Beebe

Biomedical Microdevices 2007 Volume 9( Issue 4) pp:611-617

Publication Date(Web):2007 August

DOI:10.1007/s10544-007-9074-2

There is a need for cell purification strategies suitable for handling small cell populations (tens to thousands of cells). We present here a novel strategy for small scale cell purifications using lectin-modified microchannels. Mammalian cells can be selectively captured in lectin-modified channels and eluted with a solution of the lectin’s inhibiting sugar. As a proof-of-principle demonstration, two cell lines with different binding affinities to the lectin Sambucus nigra agglutinin (SNA) were combined at various ratios and introduced into SNA-modified microchannels. After incubation and washing, enrichments of approximately ten-fold were obtained for the SNA-binding cell type. The results demonstrate the potential of this miniaturized device for manipulation and purification of limited quantities of cells.

Identification of Mammalian Cell Lines Using MALDI-TOF and LC-ESI-MS/MS Mass Spectrometry

Co-reporter:Xu Zhang, Mark Scalf, Travis W. Berggren, Michael S. Westphall, Lloyd M. Smith

Journal of the American Society for Mass Spectrometry 2006 Volume 17(Issue 4) pp:490-499

Publication Date(Web):April 2006

DOI:10.1016/j.jasms.2005.12.007

Direct mass spectrometric analysis of complex biological samples is becoming an increasingly useful technique in the field of proteomics. Matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS) is a rapid and sensitive analytical tool well suited for obtaining molecular weights of peptides and proteins from complex samples. Here, a fast and simple approach to cellular protein profiling is described in which mammalian cells are lysed directly in the MALDI matrix 2,5-dihydroxybenzoic acid (DHB) and mass analyzed using MALDI-time of flight (TOF). Using the unique MALDI mass spectral “fingerprint” generated in these analyses, it is possible to differentiate among several different mammalian cell lines. A number of techniques, including MALDI-post source decay (PSD), MALDI tandem time-of-flight (TOF-TOF), MALDI-Fourier transform ion cyclotron resonance (FTICR), and nanoflow liquid chromatography followed by electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) were employed to attempt to identify the proteins represented in the MALDI spectra. Performing a tryptic digestion of the supernatant of the cells lysed in DHB with subsequent LC-ESI-MS/MS analysis was by far the most successful method to identify proteins.

Controlling Gas-Phase Reactions for Efficient Charge Reduction Electrospray Mass Spectrometry of Intact Proteins

Co-reporter:Brian L. Frey, Yuan Lin, Michael S. Westphall, Lloyd M. Smith

Journal of the American Society for Mass Spectrometry 2005 Volume 16(Issue 11) pp:1876-1887

Publication Date(Web):November 2005

DOI:10.1016/j.jasms.2005.07.019

Charge reduction electrospray mass spectrometry (CREMS) reduces the charge states of electrospray-generated ions, which concentrates the ions from a protein into fewer peaks spread over a larger m/z range, thereby increasing peak separation and decreasing spectral congestion. An optimized design for a CREMS source is described that provides an order-of-magnitude increase in sensitivity compared to previous designs and provides control over the extent of charge reduction. Either a corona discharge or an α-particle source was employed to generate anions that abstract protons from electrosprayed protein cations. These desired ion/ion proton transfer reactions predominated, but some oxidation and ion-attachment reactions also occurred, leading to new peaks or mass-shifted broader peaks while decreasing signal intensity. The species producing these deleterious side-reactions were identified, and conditions were found that prevented their formation. Spectrometer m/z biases were examined because of their effect upon the signal intensity of higher m/z charge-reduced protein ions. The utility of this atmospheric pressure CREMS was demonstrated using a cell lysate fraction from E. coli. The spectral simplification afforded by CREMS reveals more proteins than are observed without charge reduction.

Invasive cleavage reactions on DNA-modified diamond surfaces

Co-reporter:Manchun Lu;Tanya Knickerbocker;Wei Cai;Wensha Yang;Robert J. Hamers

Biopolymers 2004 Volume 73(Issue 5) pp:

Publication Date(Web):17 FEB 2004

DOI:10.1002/bip.20007

Recently developed DNA-modified diamond surfaces exhibit excellent chemical stability to high-temperature incubations in biological buffers. The stability of these surfaces is substantially greater than that of gold or silicon surfaces, using similar surface attachment chemistry. The DNA molecules attached to the diamond surfaces are accessible to enzymes and can be modified in surface enzymatic reactions. An important application of these surfaces is for surface invasive cleavage reactions, in which target DNA strands added to the solution may result in specific cleavage of surface-bound probe oligonucleotides, permitting analysis of single nucleotide polymorphisms (SNPs). Our previous work demonstrated the feasibility of performing such cleavage reactions on planar gold surfaces using PCR-amplified human genomic DNA as target. The sensitivity of detection in this earlier work was substantially limited by a lack of stability of the gold surface employed. In the present work, detection sensitivity is improved by a factor of ∼100 (100 amole of DNA target compared with 10 fmole in the earlier work) by replacing the DNA-modified gold surface with a more stable DNA-modified diamond surface. © 2004 Wiley Periodicals, Inc. Biopolymers, 2004

DNA computing on surfaces

Co-reporter:Qinghua Liu, Liman Wang, Anthony G. Frutos, Anne E. Condon, Robert M. Corn and Lloyd M. Smith

Nature 2000 403(6766) pp:175

Publication Date(Web):

DOI:10.1038/35003155

DNA computing was proposed1 as a means of solving a class of intractable computational problems in which the computing time can grow exponentially with problem size (the ‘NP-complete’ or non-deterministic polynomial time complete problems). The principle of the technique has been demonstrated experimentally for a simple example of the hamiltonian path problem2 (in this case, finding an airline flight path between several cities, such that each city is visited only once3). DNA computational approaches to the solution of other problems have also been investigated4, 5, 6, 7, 8, 9. One technique10, 11, 12, 13 involves the immobilization and manipulation of combinatorial mixtures of DNA on a support. A set of DNA molecules encoding all candidate solutions to the computational problem of interest is synthesized and attached to the surface. Successive cycles of hybridization operations and exonuclease digestion are used to identify and eliminate those members of the set that are not solutions. Upon completion of all the multi-step cycles, the solution to the computational problem is identified using a polymerase chain reaction to amplify the remaining molecules, which are then hybridized to an addressed array. The advantages of this approach are its scalability and potential to be automated (the use of solid-phase formats simplifies the complex repetitive chemical processes, as has been demonstrated in DNA and protein synthesis14). Here we report the use of this method to solve a NP-complete problem. We consider a small example of the satisfiability problem (SAT)2, in which the values of a set of boolean variables satisfying certain logical constraints are determined.

High resolution characterization of DNA fragment ions produced by ultraviolet matrix-assisted laser desorption/ionization using linear and reflecting time-of-flight mass spectrometry

Co-reporter:Joern Krause, Mark Scalf, Lloyd M Smith

Journal of the American Society for Mass Spectrometry 1999 Volume 10(Issue 5) pp:423-429

Publication Date(Web):May 1999

DOI:10.1016/S1044-0305(99)00009-4

A mechanism for the gas phase fragmentation of DNA during the matrix-assisted laser desorption/ionization process has been proposed by ourselves and others, in which the initiating event is nucleobase protonation, followed by base loss and backbone cleavage. The proposed mechanism predicts the structures, and hence the masses, of the fragmentation products; however in earlier studies the mass resolution was insufficient to confirm the identities of these ions. In the present study a high resolution mass spectrometer equipped with a reflectron and delayed extraction capability was employed to determine the exact masses of the fragmentation products obtained from a number of short model oligonucleotide analytes. The fragment ions observed were found to have masses consistent with those predicted in the proposed mechanism. An additional species is observed in the mass spectra which was unresolved from other ions in previous studies; this species is hypothesized to correspond to the addition of water at the site of base loss from the DNA.

Modifying the Charge State Distribution of Proteins in Electrospray Ionization Mass Spectrometry by Chemical Derivatization

Co-reporter:Casey J. Krusemark, Brian L. Frey, Peter J. Belshaw, Lloyd M. Smith

Journal of the American Society for Mass Spectrometry (September 2009) Volume 20(Issue 9) pp:1617-1625

Publication Date(Web):1 September 2009

DOI:10.1016/j.jasms.2009.04.017

Is Charge Reduction in ESI Really Necessary?

Co-reporter:Lloyd M. Smith

Journal of the American Society for Mass Spectrometry (May 2008) Volume 19(Issue 5) pp:629-631

Publication Date(Web):1 May 2008

DOI:10.1016/j.jasms.2008.02.002

The technique of charge reduction electrospray mass spectrometry (CREMS), which can reduce the charge state complexity produced in electrospray ionization (ESI), is discussed.