A common approach to isolate surface proteins from fungal and bacterial cells is to perform a proteolytic cleavage of proteins on the surface of intact cells suspended in solution. This paper describes miniaturization of this technique, in which cells are adhered on glass surfaces, and all sample treatments are conducted at <i>μ</i>L volumes. Specifically,<i> Candida albicans</i> cells were attached onto HSA-coated glass slides. By depositing the appropriate reagent solutions on the adhered cells, we successfully performed cell washing, treatment with antifugal peptide, Histatin 5, and a proteolysis on intact cells with trypsin. The resulting peptides were subsequently analysed by mass spectrometry. In general, the data obtained was similar to that collected with suspended cells in much larger sample volumes. However, our miniaturized workflow offers the benefit of greatly reducing the consumption of cells and reagents.

Shirley Fan

Eduardo B. Moffa

Yizhi Xiao

Walter L. Siqueira

Ken K.-C. Yeung

Miniaturized Digestion and Extraction of Surface Proteins from<i> Candida albicans</i> following Treatment with Histatin 5 for Mass Spectrometry Analysis

The aim of this study is to analyze the differential proteins in MRSA ATCC 33591 treated with aqueous extract from<i> Q. infectoria</i> gall. Protein extracts were obtained from MRSA cells by sonication and were separated by 2D polyacrylamide gels. Protein spots of interest were extracted from the gels and identified using LC-ESI-QTOF MS. The concentration of<i> Q. infectoria</i> extract used for 2D-gel electrophoresis was subinhibitory concentration. Minimum inhibitory concentration (MIC) value of the extract against MRSA was 19.50 <i>μ</i>g/mL with bacteriostatic action at 1x MIC from time-kill assay. However, the extract exhibited dose-dependent manner and was bactericidal at 4x MIC with more than 3 log<sub>10</sub> CFU/mL reduction at 4 h. 2D-GE map showed that 18 protein spots were upregulated and another six were downregulated more than twofold (<svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-3.42938pt" id="M1" height="11.7782pt" version="1.1" viewBox="-0.0498162 -8.34882 44.5261 11.7782" width="44.5261pt"><g transform="matrix(.013,0,0,-0.013,0,0)"><path id="g113-113" d="M570 304C570 398 525 448 414 448C385 448 343 445 312 434L329 511L321 518C297 504 262 482 244 460L233 411C195 397 159 381 128 358L135 332C160 347 189 360 224 373L111 -147C97 -210 84 -218 17 -231L13 -257L254 -247L259 -218L233 -216C183 -212 177 -202 189 -142L218 -1C238 -10 266 -12 283 -12C351 3 429 48 483 105C543 168 570 242 570 304ZM482 289C482 161 380 33 304 33C278 33 248 51 233 69L303 396C326 400 352 403 369 403C428 403 482 380 482 289Z"/></g><g transform="matrix(.013,0,0,-0.013,11.342,0)"><path id="g117-91" d="M512 -3V55L134 254V256L512 456V514L75 281V230L512 -3Z"/></g><g transform="matrix(.013,0,0,-0.013,22.605,0)"><path id="g113-49" d="M241 635C89 635 35 457 35 312C35 153 89 -12 240 -12C390 -12 443 166 443 312C443 466 390 635 241 635ZM238 602C329 602 354 454 354 312C354 172 330 22 240 22C152 22 124 173 124 313S148 602 238 602Z"/></g><g transform="matrix(.013,0,0,-0.013,28.845,0)"><path id="g113-47" d="M113 -12C146 -12 170 11 170 46C170 78 146 103 114 103S58 78 58 46C58 11 82 -12 113 -12Z"/></g><g transform="matrix(.013,0,0,-0.013,31.809,0)"><use xlink:href="#g113-49"/></g><g transform="matrix(.013,0,0,-0.013,38.049,0)"><path id="g113-54" d="M153 550H386L412 615L406 623H120L82 318C104 327 142 338 184 338C294 338 347 275 347 187C347 112 305 39 221 39C160 39 119 71 97 89C88 97 80 96 71 90C59 80 50 67 49 57C48 45 52 36 66 23C80 9 123 -12 169 -12C221 -11 288 15 342 59C403 109 431 165 431 225C431 308 366 395 238 395C212 395 165 379 127 364L153 550Z"/></g></svg>) after treatment with subinhibitory concentration. Out of six proteins being downregulated, four proteins were identified as ferritin and catalase, branched-chain alpha-keto acid dehydrogenase subunit E2, and succinyl-CoA ligase [ADP-forming] subunit beta. Seven upregulated proteins which have been successfully identified were 3-hydroxyacyl-CoA dehydrogenase, NAD binding domain protein, formate C-acetyltransferase, 3-hydroxyacyl-[acyl-carrier-protein] dehydratase FabZ, NAD dependent epimerase/dehydratase family protein, and phosphopantothenoyl cysteine decarboxylase. It is postulated that the main mechanism of aqueous extract from gall of<i> Q. infectoria</i> was most likely involved in energy metabolism and protein stress. 

Radhiah Khairon

Noraziah Mohamad Zin

Mariati Abdul Rahman

Dayang Fredalina Basri

Comparative Proteomic Analysis of Differential Proteins in Response to Aqueous Extract of <i>Quercus infectoria</i> Gall in Methicillin-Resistant <i>Staphylococcus aureus</i>

Urea based protein extraction of formalin-fixed paraffin-embedded (FFPE) tissue provides the most efficient workflow for proteomics due to its compatibility with liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). This study optimizes the use of urea for proteomic analysis of clinical FFPE tissue. A series of protein extraction conditions manipulating temperature and buffer composition were compared to reduce carbamylation introduced by urea and increase protein detection. Each extraction was performed on a randomized pair of serial sections of homogenous FFPE tissue and analyzed with LC-ESI-MS/MS. Results were compared in terms of yield, missed cleavages, and peptide carbamylation. Lowering extraction temperature to 60&#xb0;C decreased carbamylation at the cost of decreased protein detection and yield. Protein extraction for at least 20 minutes at 95&#xb0;C followed by 60&#xb0;C for 2 hours maximized total protein yield while maintaining protein detection and reducing carbamylation by 7.9&#x25;. When accounting for carbamylation during analysis, this modified extraction temperature provides equivalent peptide and protein detection relative to the commercially available Qproteome&#xae; FFPE Tissue Kit. No changes to buffer composition containing 7&#x2009;M urea, 2&#x2009;M thiourea, and 1&#x2009;M ammonium bicarbonate resulted in improvements to control conditions. Optimized urea in-solution digestion provides an efficient workflow with maximized yields for proteomic analysis of clinically relevant FFPE tissue.

Stephen A. Luebker

Scott A. Koepsell

Optimization of Urea Based Protein Extraction from Formalin-Fixed Paraffin-Embedded Tissue for Shotgun Proteomics

Nitric oxide (NO) protects the heart against ischemic injury; however, NO- and superoxide-dependent S-nitrosylation (S-NO) of cysteines can affect function of target proteins and play a role in disease outcome. We employed 2D-GE with thiol-labeling FL-maleimide dye and MALDI-TOF MS/MS to capture the quantitative changes in abundance and S-NO proteome of HF patients (versus healthy controls, <svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-0.2063904pt" id="M1" height="8.55521pt" version="1.1" viewBox="-0.0498162 -8.34882 34.1002 8.55521" width="34.1002pt"><g transform="matrix(.013,0,0,-0.013,0,0)"><path id="g113-111" d="M495 86L479 114C446 82 419 66 409 66C401 66 401 72 406 97C420 166 436 231 453 297C489 435 454 448 428 448C406 448 384 439 354 422C305 394 222 327 161 247H159L183 345C200 415 194 448 173 448C143 448 82 410 23 351L38 325C64 349 95 371 105 371C111 371 116 365 109 336L25 -4L31 -12C50 -4 77 3 107 9C119 69 132 122 145 168C197 254 321 381 370 381C387 381 393 374 378 305L329 95C309 17 320 -12 345 -12C372 -12 430 19 495 86Z"/></g><g transform="matrix(.013,0,0,-0.013,10.158,0)"><path id="g117-34" d="M535 323V373H52V323H535ZM535 138V188H52V138H535Z"/></g><g transform="matrix(.013,0,0,-0.013,21.421,0)"><path id="g113-52" d="M285 378C315 398 338 416 353 432C373 451 384 474 384 503C384 579 325 635 236 635H235C182 635 136 610 108 579L65 516L85 496C110 533 150 575 205 575C258 575 300 543 300 481C300 407 232 369 141 339L147 310C163 315 188 321 211 321C268 321 338 284 338 192C338 94 288 40 217 40C160 40 119 68 93 91C85 98 77 97 69 91C60 84 47 71 46 58C44 46 48 35 62 22C75 10 116 -12 162 -12C234 -12 424 62 424 224C424 297 373 359 285 376V378Z"/></g><g transform="matrix(.013,0,0,-0.013,27.661,0)"><path id="g113-49" d="M241 635C89 635 35 457 35 312C35 153 89 -12 240 -12C390 -12 443 166 443 312C443 466 390 635 241 635ZM238 602C329 602 354 454 354 312C354 172 330 22 240 22C152 22 124 173 124 313S148 602 238 602Z"/></g></svg>/group). We identified 93 differentially abundant (59-increased/34-decreased) and 111 S-NO-modified (63-increased/48-decreased) protein spots, respectively, in HF subjects (versus controls, fold-change <svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-2.15067pt" id="M2" height="11.439pt" version="1.1" viewBox="-0.0498162 -9.28833 4.25782 11.439" width="4.25782pt"><g transform="matrix(.013,0,0,-0.013,0,0)"><path id="g113-9" d="M162 -163V703H101V-163H162Z"/></g></svg>≥1.5<svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-2.15067pt" id="M3" height="11.439pt" version="1.1" viewBox="-0.0498162 -9.28833 3.53145 11.439" width="3.53145pt"><g transform="matrix(.013,0,0,-0.013,0,0)"><use xlink:href="#g113-9"/></g></svg>, <svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-3.42938pt" id="M4" height="11.7782pt" version="1.1" viewBox="-0.0498162 -8.34882 44.5261 11.7782" width="44.5261pt"><g transform="matrix(.013,0,0,-0.013,0,0)"><path id="g113-113" d="M570 304C570 398 525 448 414 448C385 448 343 445 312 434L329 511L321 518C297 504 262 482 244 460L233 411C195 397 159 381 128 358L135 332C160 347 189 360 224 373L111 -147C97 -210 84 -218 17 -231L13 -257L254 -247L259 -218L233 -216C183 -212 177 -202 189 -142L218 -1C238 -10 266 -12 283 -12C351 3 429 48 483 105C543 168 570 242 570 304ZM482 289C482 161 380 33 304 33C278 33 248 51 233 69L303 396C326 400 352 403 369 403C428 403 482 380 482 289Z"/></g><g transform="matrix(.013,0,0,-0.013,11.342,0)"><path id="g117-93" d="M531 71V127L115 310L531 494V550L57 335V285L531 71ZM531 -40V10H57V-40H531Z"/></g><g transform="matrix(.013,0,0,-0.013,22.605,0)"><use xlink:href="#g113-49"/></g><g transform="matrix(.013,0,0,-0.013,28.845,0)"><path id="g113-47" d="M113 -12C146 -12 170 11 170 46C170 78 146 103 114 103S58 78 58 46C58 11 82 -12 113 -12Z"/></g><g transform="matrix(.013,0,0,-0.013,31.809,0)"><use xlink:href="#g113-49"/></g><g transform="matrix(.013,0,0,-0.013,38.049,0)"><path id="g113-54" d="M153 550H386L412 615L406 623H120L82 318C104 327 142 338 184 338C294 338 347 275 347 187C347 112 305 39 221 39C160 39 119 71 97 89C88 97 80 96 71 90C59 80 50 67 49 57C48 45 52 36 66 23C80 9 123 -12 169 -12C221 -11 288 15 342 59C403 109 431 165 431 225C431 308 366 395 238 395C212 395 165 379 127 364L153 550Z"/></g></svg>). Ingenuity pathway analysis of proteome datasets suggested that the pathways involved in phagocytes’ migration, free radical production, and cell death were activated and fatty acid metabolism was decreased in HF subjects. Multivariate adaptive regression splines modeling of datasets identified a panel of proteins that will provide &gt;90% prediction success in classifying HF subjects. Proteomic profiling identified ATP-synthase, thrombospondin-1 (THBS1), and vinculin (VCL) as top differentially abundant and S-NO-modified proteins, and these proteins were verified by Western blotting and ELISA in different set of HF subjects. We conclude that differential abundance and S-NO modification of proteins serve as a mechanism in regulating cell viability and free radical production, and THBS1 and VCL evaluation will potentially be useful in the prediction of heart failure. 

Sue-jie Koo

Heidi M. Spratt

Kizhake V. Soman

Susan Stafford

Shivali Gupta

John R. Petersen

Maria P. Zago

Muge N. Kuyumcu-Martinez

Allan R. Brasier

John E. Wiktorowicz

Nisha Jain Garg

S-Nitrosylation Proteome Profile of Peripheral Blood Mononuclear Cells in Human Heart Failure

Yeast flavohemoglobin, YHb, encoded by the nuclear gene YHB1, has been implicated in the nitrosative stress responses in<i> Saccharomyces cerevisiae</i>. It is still unclear how<i> S. cerevisiae</i> can withstand this NO level in the absence of flavohemoglobin. To better understand the physiological function of flavohemoglobin in yeast, in the present study a label-free differential proteomics study has been carried out in wild-type and YHB1 deleted strains of<i> S. cerevisiae</i> grown under fermentative conditions. From the analysis, 417 proteins in Y190 and 392 proteins in ΔYHB1 were identified with high confidence. Interestingly, among the differentially expressed identified proteins, 40 proteins were found to be downregulated whereas 41 were found to be upregulated in ΔYHB1 strain of<i> S. cerevisiae</i> (<svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-3.42943pt" id="M1" height="10.2124pt" version="1.1" viewBox="-0.0498162 -6.78297 7.83752 10.2124" width="7.83752pt"><g transform="matrix(.013,0,0,-0.013,0,0)"><path id="g113-113" d="M570 304C570 398 525 448 414 448C385 448 343 445 312 434L329 511L321 518C297 504 262 482 244 460L233 411C195 397 159 381 128 358L135 332C160 347 189 360 224 373L111 -147C97 -210 84 -218 17 -231L13 -257L254 -247L259 -218L233 -216C183 -212 177 -202 189 -142L218 -1C238 -10 266 -12 283 -12C351 3 429 48 483 105C543 168 570 242 570 304ZM482 289C482 161 380 33 304 33C278 33 248 51 233 69L303 396C326 400 352 403 369 403C428 403 482 380 482 289Z"/></g></svg> value &lt; 0.05)<i>.</i> The differentially expressed proteins were also classified according to gene ontology (GO) terms. The most enriched and significant GO terms included nitrogen compound biosynthesis, amino acid biosynthesis, translational regulation, and protein folding. Interactions of differentially expressed proteins were generated using Search Tool for the Retrieval of Interacting Genes (STRING) database. This is the first report which offers a more complete view of the proteome changes in<i> S. cerevisiae</i> in the absence of flavohemoglobin. 

Chiranjit Panja

Rakesh K.S. Setty

Gopal Vaidyanathan

Sanjay Ghosh

Label-Free Proteomic Analysis of Flavohemoglobin Deleted Strain of<i> Saccharomyces cerevisiae</i>

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International Journal of Proteomics

Table of Contents

Table of Contents: 2016

Miniaturized Digestion and Extraction of Surface Proteins from Candida albicans following Treatment with Histatin 5 for Mass Spectrometry Analysis

Comparative Proteomic Analysis of Differential Proteins in Response to Aqueous Extract of Quercus infectoria Gall in Methicillin-Resistant Staphylococcus aureus

Optimization of Urea Based Protein Extraction from Formalin-Fixed Paraffin-Embedded Tissue for Shotgun Proteomics

S-Nitrosylation Proteome Profile of Peripheral Blood Mononuclear Cells in Human Heart Failure

Label-Free Proteomic Analysis of Flavohemoglobin Deleted Strain of Saccharomyces cerevisiae